New Drug Development

New Drug
Development
Copyright © 2004 by Marcel Dekker, Inc.

DRUGS AND THE PHARMACEUTICAL SCIENCES
Executive Editor
James Swarbrick
PharmaceuTech, Inc.
Pinehurst, North Carolina
Advisory Board
Larry L.Augsburger Harry G.Brittain

University of Maryland Center for Pharmaceutical Physics
Baltimore, Maryland Milford, New Jersey
Jennifer B.Dressman Anthony J.Mickey

Johann Wolfgang Goethe-University University of North Carolina School of
Frankfurt, Germany Pharmacy
Chapel Hill, North Carolina
Jeffrey A.Hughes
University of Florida College of Pharmacy Ajaz Hussain
Gainesville, Florida U.S. Food and Drug Administration
Frederick, Maryland
Trevor M.Jones
The Association of the Hans E.Junginger
British Pharmaceutical Industry Leiden/Amsterdam Center for Drug Research
London, United Kingdom Leiden, The Netherlands
Vincent H.L.Lee Stephen G.Schulman

University of Southern California University of Florida
Los Angeles, California Gainesville, Florida
Jerome P.Skelly Elizabeth M.Topp

Alexandria, Virginia University of Kansas School of Pharmacy
Lawrence, Kansas
Geoffrey T.Tucker
University of Sheffield Peter York
Royal Hallamshire Hospital University of Bradford School of Pharmacy
Sheffield, United Kingdom Bradford, United Kingdom

DRUGS AND THE PHARMACEUTICAL SCIENCES
A Series of Textbooks and Monographs
1. Pharmacokinetics, Milo Gibaldi and Donald Perrier

2. Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality
Control, Sidney H.Willig, Murray M.Tuckerman, and William S.Hitchings IV
3. Microencapsulatlon, edited by J.R.Nixon
4. Drug Metabolism: Chemical and Biochemical Aspects, Bernard Testa and
Peter Jenner
5. New Drugs: Discovery and Development, edited by Alan A.Rubin
6. Sustained and Controlled Release Drug Delivery Systems, edited by Joseph
R.Robinson
7. Modern Pharmaceutics, edited by Gilbert S.Banker and Christopher T.Rhodes
8. Prescription Drugs in Short Supply: Case Histories, Michael A.Schwartz
9. Activated Charcoal: Antidotal and Other Medical Uses, David O.Cooney
10. Concepts in Drug Metabolism (in two parts), edited by Peter Jenner and
Bernard Testa
11. Pharmaceutical Analysis: Modern Methods (in two parts), edited by James
W.Munson
12. Techniques of Solubilization of Drugs, edited by Samuel H.Yalkowsky
13. Orphan Drugs, edited by Fred E.Karch
14. Novel Drug Delivery Systems: Fundamentals, Developmental Concepts,
Biomedical Assessments, Yie W.Chien
15. Pharmacokinetics: Second Edition, Revised and Expanded, Milo Gibaldi and
Donald Perrier
Control, Second Edition, Revised and Expanded, Sidney H.Willig, Murray
M.Tuckerman, and William S.Hitchings IV
17. Formulation of Veterinary Dosage Forms, edited by Jack Blodinger
18. Dermatological Formulations: Percutaneous Absorption, Brian W.Barry
19. The Clinical Research Process in the Pharmaceutical Industry, edited by Gary
M.Matoren
20. Microencapsulation and Related Drug Processes, Patrick B.Deasy
21. Drugs and Nutrients: The Interactive Effects, edited by Daphne A.Roe and
T.Colin Campbell
22. Biotechnology of Industrial Antibiotics, Erick J.Vandamme

23. Pharmaceutical Process Validation, edited by Bernard T.Loftus and Robert
A.Nash
24. Anticancer and Interferon Agents: Synthesis and Properties, edited by
Raphael M.Ottenbrite and George B.Butler
25. Pharmaceutical Statistics: Practical and Clinical Applications, Sanford Bolton
26. Drug Dynamics for Analytical, Clinical, and Biological Chemists, Benjamin
J.Gudzinowicz, Burrows T.Younkin, Jr., and Michael J.Gudzinowicz
27. Modern Analysis of Antibiotics, edited by Adjoran Aszalos
28. Solubility and Related Properties, Kenneth C.James
29. Controlled Drug Delivery: Fundamentals and Applications, Second Edition,
Revised and Expanded, edited by Joseph R.Robinson and Vincent H.Lee
30. New Drug Approval Process: Clinical and Regulatory Management, edited by
Richard A.Guarino
31. Transdermal Controlled Systemic Medications, edited by Yie W.Chien
32. Drug Delivery Devices: Fundamentals and Applications, edited by Praveen
Tyle
33. Pharmacokinetics: Regulatory • Industrial • Academic Perspectives, edited by
Peter G.Welling and Francis L S.Tse
34. Clinical Drug Trials and Tribulations, edited by Allen E.Cato
35. Transdermal Drug Delivery: Developmental Issues and Research Initiatives,
edited by Jonathan Hadgraft and Richard H.Guy
36. Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms, edited by
James W.McGinity
37. Pharmaceutical Pelletization Technology, edited by Isaac Ghebre-Sellassie
38. Good Laboratory Practice Regulations, edited by Allen F.Hirsch
39. Nasal Systemic Drug Delivery, Yie W.Chien, Kenneth S.E.Su, and Shyi-Feu
Chang
40. Modern Pharmaceutics: Second Edition, Revised and Expanded, edited by
Gilbert S.Banker and Christopher T.Rhodes
41. Specialized Drug Delivery Systems: Manufacturing and Production
Technology, edited by Praveen Tyle
42. Topical Drug Delivery Formulations, edited by David W.Osborne and Anton
H.Amann
43. Drug Stability: Principles and Practices, Jens T.Carstensen
44. Pharmaceutical Statistics: Practical and Clinical Applications, Second Edition,
Revised and Expanded, Sanford Bolton
45. Biodegradable Polymers as Drug Delivery Systems, edited by Mark Chasin
and Robert Langer
46. Preclinical Drug Disposition: A Laboratory Handbook, Francis L S.Tse and
James J.Jaffe
47. HPLC in the Pharmaceutical Industry, edited by Godwin W.Fong and Stanley
K.Lam
48. Pharmaceutical Bioequivalence, edited by Peter G.Welling, Francis L S.Tse,
and Shrikant V.Dinghe

49. Pharmaceutical Dissolution Testing, Umesh V. Banakcar
50. Novel Drug Delivery Systems: Second Edition, Revised and Expanded, Yie
W.Chien
51. Managing the Clinical Drug Development Process, David M.Cocchetto and
Ronald V.Nardi
Control, Third Edition, edited by Sidney H.Willig and James R.Stoker
53. Prodrugs: Topical and Ocular Drug Delivery, edited by Kenneth B. Sloan
54. Pharmaceutical Inhalation Aerosol Technology, edited by Anthony J.Hickey
55. Radiopharmaceuticals: Chemistry and Pharmacology, edited by Adrian
D.Nunn
56. New Drug Approval Process: Second Edition, Revised and Expanded, edited
by Richard A.Guarino
57. Pharmaceutical Process Validation: Second Edition, Revised and Expanded,
edited by Ira R.Berry and Robert A.Nash
58. Ophthalmic Drug Delivery Systems, edited by Ashim K.Mitra
59. Pharmaceutical Skin Penetration Enhancement, edited by Kenneth A.Walters
and Jonathan Hadgraft
60. Colonic Drug Absorption and Metabolism, edited by Peter R.Bieck
61. Pharmaceutical Particulate Carriers: Therapeutic Applications, edited by Alain
Rolland
62. Drug Permeation Enhancement: Theory and Applications, edited by Dean
S.Hsieh
63. Glycopeptide Antibiotics, edited by Ramakrishnan Nagarajan
64. Achieving Sterility in Medical and Pharmaceutical Products, Nigel A.Halls
65. Multiparticulate Oral Drug Delivery, edited by Isaac Ghebre-Sellassie
66. Colloidal Drug Delivery Systems, edited by Jörg Kreuter
67. Pharmacokinetics: Regulatory • Industrial • Academic Perspectives, Second
Edition, edited by Peter G.Welling and Francis L S.Tse
68. Drug Stability: Principles and Practices, Second Edition, Revised and
Expanded, Jens T.Carstensen
69. Good Laboratory Practice Regulations: Second Edition, Revised and
Expanded, edited by Sandy Weinberg
70. Physical Characterization of Pharmaceutical Solids, edited by Harry G.
Brittain
71. Pharmaceutical Powder Compaction Technology, edited by Göran Alderborn
and Christer Nyström
72. Modern Pharmaceutics: Third Edition, Revised and Expanded, edited by
73. Microencapsulation: Methods and Industrial Applications, edited by Simon
Benita
74. Oral Mucosal Drug Delivery, edited by Michael J.Rathbone
75. Clinical Research in Pharmaceutical Development, edited by Barry Bleidt and
Michael Montagne

76. The Drug Development Process: Increasing Efficiency and Cost Effectiveness,
edited by Peter G.Welling, Louis Lasagna, and Umesh V.Banakar
77. Microparticulate Systems for the Delivery of Proteins and Vaccines, edited by
Smadar Cohen and Howard Bernstein
Control, Fourth Edition, Revised and Expanded, Sidney H.Willig and James
R.Stoker
79. Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms: Second
Edition, Revised and Expanded, edited by James W.McGinity
80. Pharmaceutical Statistics: Practical and Clinical Applications, Third Edition,
Sanford Bolton
81. Handbook of Pharmaceutical Granulation Technology, edited by Dilip M.Parikh
82. Biotechnology of Antibiotics: Second Edition, Revised and Expanded, edited
by William R.Strohl
83. Mechanisms of Transdermal Drug Delivery, edited by Russell O.Potts and
Richard H.Guy
84. Pharmaceutical Enzymes, edited by Albert Lauwers and Simon Scharpé
85. Development of Biopharmaceutical Parenteral Dosage Forms, edited by John
A.Bontempo
86. Pharmaceutical Project Management, edited by Tony Kennedy
87. Drug Products for Clinical Trials: An International Guide to Formulation •
Production • Quality Control, edited by Donald C.Monkhouse and Christopher
T.Rhodes
88. Development and Formulation of Veterinary Dosage Forms: Second Edition,
Revised and Expanded, edited by Gregory E.Hardee and J.Desmond Baggot
89. Receptor-Based Drug Design, edited by Paul Left
90. Automation and Validation of Information in Pharmaceutical Processing,
edited by Joseph F.deSpautz
91. Dermal Absorption and Toxicity Assessment, edited by Michael S.Roberts and
Kenneth A.Walters
92. Pharmaceutical Experimental Design, Gareth A.Lewis, Didier Mathieu, and
Roger Phan-Tan-Luu
93. Preparing for FDA Pre-Approval Inspections, edited by Martin D.Hynes III
94. Pharmaceutical Excipients: Characterization by IR, Raman, and NMR
Spectroscopy, David E.Bugay and W.Paul Findlay
95. Polymorphism in Pharmaceutical Solids, edited by Harry G Brittain
96. Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products,
edited by Louis Rey and Joan C.May
97. Percutaneous Absorption: Drugs-Cosmetics-Mechanisms-Methodology, Third
Edition, Revised and Expanded, edited by Robert L.Bronaugh and Howard
L.Maibach
98. Bioadhesive Drug Delivery Systems: Fundamentals, Novel Approaches, and
Development, edited by Edith Mathiowitz, Donald E.Chickering III, and Claus-
Michael Lehr

99. Protein Formulation and Delivery, edited by Eugene J.McNally
100. New Drug Approval Process: Third Edition, The Global Challenge, edited by
Richard A.Guarino
101. Peptide and Protein Drug Analysis, edited by Ronald E.Reid
102. Transport Processes in Pharmaceutical Systems, edited by Gordon L.
Amidon, Ping I.Lee, and Elizabeth M.Topp
103. Excipient Toxicity and Safety, edited by Myra L.Weiner and Lois A.Kotkoskie
104. The Clinical Audit in Pharmaceutical Development, edited by Michael
R.Hamrell
105. Pharmaceutical Emulsions and Suspensions, edited by Francoise Nielloud
and Gilberte Marti-Mestres
106. Oral Drug Absorption: Prediction and Assessment, edited by Jennifer
B.Dressman and Hans Lennernäs
107. Drug Stability: Principles and Practices, Third Edition, Revised and
Expanded, edited by Jens T.Carstensen and C.T.Rhodes
108. Containment in the Pharmaceutical Industry, edited by James P.Wood
Control from Manufacturer to Consumer, Fifth Edition, Revised and
Expanded, Sidney H.Willig
110. Advanced Pharmaceutical Solids, Jens T.Carstensen
111. Endotoxins: Pyrogens, LAL Testing, and Depyrogenation, Second Edition,
Revised and Expanded, Kevin L. Williams
112. Pharmaceutical Process Engineering, Anthony J.Mickey and David
Ganderton
113. Pharmacogenomics, edited by Werner Kalow, Urs A.Meyer, and Rachel
F.Tyndale
114. Handbook of Drug Screening, edited by Ramakrishna Seethala and
Prabhavathi B.Fernandes
115. Drug Targeting Technology: Physical • Chemical • Biological Methods, edited
by Hans Schreier
116. Drug-Drug Interactions, edited by A.David Rodrigues
117. Handbook of Pharmaceutical Analysis, edited by Lena Ohannesian and
Anthony J.Streeter
118. Pharmaceutical Process Scale-Up, edited by Michael Levin
119. Dermatological and Transdermal Formulations, edited by Kenneth A.
Walters
120. Clinical Drug Trials and Tribulations: Second Edition, Revised and Expanded,
edited by Allen Cato, Lynda Sutton, and Allen Cato III
121. Modern Pharmaceutics: Fourth Edition, Revised and Expanded, edited by
122. Surfactants and Polymers in Drug Delivery, Martin Malmsten
123. Transdermal Drug Delivery: Second Edition, Revised and Expanded, edited
by Richard H.Guy and Jonathan Hadgraft
124. Good Laboratory Practice Regulations: Second Edition, Revised and
Expanded, edited by Sandy Weinberg
125. Parenteral Quality Control: Sterility, Pyrogen, Particulate, and Package

Integrity Testing: Third Edition, Revised and Expanded, Michael J.Akers,
Daniel S.Larrimore, and Dana Morton Guazzo
126. Modified-Release Drug Delivery Technology, edited by Michael J.Rathbone,
Jonathan Hadgraft, and Michael S.Roberts
127. Simulation for Designing Clinical Trials: A Pharmacokinetic-Pharma-
codynamic Modeling Perspective, edited by Hui C.Kimko and Stephen
B.Duffull
128. Affinity Capillary Electrophoresis in Pharmaceutics and Biopharmaceutics,
edited by Reinhard H.H.Neubert and Hans-Hermann Rüttinger
129. Pharmaceutical Process Validation: An International Third Edition, Revised
and Expanded, edited by Robert A.Nash and Alfred H.Wachter
130. Ophthalmic Drug Delivery Systems: Second Edition, Revised and Expanded,
edited by Ashim K.Mitra
131. Pharmaceutical Gene Delivery Systems, edited by Alain Rolland and Sean
M.Sullivan
132. Biomarkers in Clinical Drug Development, edited by John C.Bloom and
Robert A.Dean
133. Pharmaceutical Extrusion Technology, edited by Isaac Ghebre-Sellassie and
Charles Martin
134. Pharmaceutical Inhalation Aerosol Technology: Second Edition, Revised and
Expanded, edited by Anthony J.Hickey
135. Pharmaceutical Statistics: Practical and Clinical Applications, Fourth Edition,
Sanford Bolton and Charles Bon
136. Compliance Handbook for Pharmaceuticals, Medical Devices, and Biologies,
edited by Carmen Medina
137. Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products:
Second Edition, Revised and Expanded, edited by Louis Rey and Joan
C.May
138. Supercritical Fluid Technology for Drug Product Development, edited by
Peter York, Uday B.Kompella, and Boris Y.Shekunov
139. New Drug Approval Process: Fourth Edition, Accelerating Global
Registrations, edited by Richard A.Guarino
140. Microbial Contamination Control in Parenteral Manufacturing, edited by
Kevin L.Williams
141. New Drug Development: Regulatory Paradigms for Clinical Pharmacology
and Biopharmaceutics, edited by Chandrahas G.Sahajwalla
142. Microbial Contamination Control in the Pharmaceutical Industry, edited by
Luis Jimenez
ADDITIONAL VOLUMES IN PREPARATION
Generic Drug Development: Solid Oral Dosage Forms, edited by Leon

Shargel and Izzy Kanfer

Introduction to the Pharmaceutical Regulatory Process, edited by Ira R.Berry
Drug Delivery to the Oral Cavity: Molecules to Market, edited by Tapash

Ghosh and William R.Pfister

New Drug
Development
Regulatory Paradigms for Clinical Pharmacology
and Biopharmaceutics
edited by
Chandrahas G.Sahajwalla
U.S. Food and Drug Administration
Rockville, Maryland, U.S.A.
MARCEL DEKKER, INC. NEW YORK • BASEL

The views expressed in this book are those of the author’s and do not reflect the
official policy of the FDA. No official support or endorsement by the FDA is
intended or should be inferred.
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neither the author(s) nor the publisher, nor anyone else associated with this
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caused or alleged to be caused by this book. The material contained herein is not
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Trademark notice: Product or corporate names may be trademarks or registered

trademarks and are used only for identification and explanation without intent to
infringe.
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Copyright © 2004 by Marcel Dekker, Inc. All Rights Reserved.
Neither this book nor any part may be reproduced or transmitted in any form or by
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Current Printing (last digit):
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PRINTED IN THE UNITED STATES OF AMERICA

With affection and appreciation
to Sri Sathya Sai Baba, for his love and guidance;
to my parents, Gope K.Sahajwalla and late Kamala G.Sahajwalla, for

teaching me the right human values;
to my mother-in-law, Devi Chawla, for her love and blessings;
to my wife, Maya, for her support, encouragement, editorial help and

critique;
to my son, Aditya, and daughter, Divya, for their unconditional and

eternal love, and bringing joy and bliss in our family.

Foreword
The opportunity to contribute to people’s health is a source of inspiration to

those working in drug development. However, drug development is
complex, costly, and fraught with uncertainty. Success demands teamwork
and extensive knowledge of current technology and regulations. The
discipline of clinical pharmacology has, over the years, become an
important and integral part of the drug development process. Now, in the
era of individualization of drug therapies, the discipline of clinical
pharmacology is strategically positioned to make seminal contributions to
the understanding of the sources of variability in individual drug responses.
The biomedical advances of recent years have the potential to transform
the drug development process; however, this goal can only be achieved if
knowledgeable people from industry, academia, and government work
together as a team. It is important that scientific personnel involved in drug
development have access to up-to-date information. New Drug
Development: Regulatory Paradigms for Clinical Pharmacology, edited by
Chandrahas Sahajwalla, is a timely book which combines the scientific and
regulatory aspects of clinical pharmacology and biopharmaceutics in easy-
to-understand chapters that cover all aspects of drug development for these
disciplines. For universities offering programs in drug development, this
volume fills an existing void, and further provides a quick reference guide
for the industrial or academic scientist who is new in the field of drug
development.
Until now there has been no specific source where a student or new
investigator could find a single, comprehensive presentation of the scientific
and regulatory principles necessary for filing the clinical pharmacology and
biopharmaceutics section of a new drug application (NDA) or biologies
v
vi Foreword
license application (BLA). Although this information is available in a

fragmentary manner in multiple places, there has been no concise reference
that gives a complete overview of the scientific and regulatory perspective
and paradigms for clinical pharmacology and biopharmaceutics.
New Drug Development: Regulatory Paradigms for Clinical
Pharmacology is unique in that it covers the regulations governing
Investigational New Drugs (IND) and NDAs, and takes the reader through
the pertinent aspects of clinical pharmacology and biopharmaceutics. This
book covers in-vitro studies needed to understand properties of new drug
molecules including metabolism, transporters, and interaction studies. Also
included are basic concepts of bioavailability and bioequivalence, specific
population studies including those in disease states such as renal and hepatic
impairment, biomarkers, population pharmacokinetics, exposure-response
studies, drug interactions and specific scientific issues related to selected
therapeutic areas. There is also very timely coverage of specific drug
development issues for chiral drugs, liposomal products, counter-
bioterrorism agents, and the regulation of antidotes for nerve agent
poisoning. Essential elements of biopharmaceutics for new and generic
drugs have also been discussed in detail.
The contributing authors are well recognized experts in their respective
fields who bring experience from regulatory organizations and academia. A
global perspective is provided by the participation of authors from Europe,
Canada, and the United States.
Rising prescription costs worldwide call for a reduction in drug
development costs whenever possible. This can be facilitated by access to
good information to assist developers in reducing the number of
unnecessary or poorly designed studies. New Drug Development:
Regulatory Paradigms for Clinical Pharmacology will provide solid
information to students, teachers, and new researchers alike and can also
serve as a quick reference for particular aspects of clinical pharmacology
and biopharmaceutics for experienced scientists.
Janet Woodcock, M.D.

Center Director
Center for Drug Evaluation and Research
Food and Drug Administration

Preface
After graduating in pharmaceutics and joining a multinational

pharmaceutical company, I quickly realized how much I need to learn about
drug development and the associated regulatory process. Most
pharmaceutical scientists have gained knowledge of regulatory science from
practical experience. There is not a single textbook that combines scientific
and regulatory principles essential to answering the clinical pharmacology
and biopharmecutics questions that arise during drug development.
Motivated by the lack of such a book, I compiled this text. This book is
aimed at students and new scientists in the industry and government, and at
encouraging universities to incorporate training for regulatory sciences in
their curriculum.
This book has been divided into five parts: History and Basic Principles
(Chapters 1–4); In Vitro/Pre-Clinical (Chapters 5–7); Clinical
Pharmacology (Chapters 8–16); Biopharmaceutics (Chapters 17–20) and
Contemporary and Special Interest Topics (Chapters 21–25).
The first part of this book introduces the reader to regulatory history,
important regulations governing clinical pharmacology and
biopharmaceutics portion of the new drug application, and the review
process at the Food and Drug Administration (FDA). This is followed by a
part in-vitro and preclinical studies such as metabolism, drug-drug
interactions, transporters and interspecies scaling. Part III introduces the
reader to clinical pharmacology studies that are generally conducted. This
part starts with a chapter on analytical method validation, and takes the
reader through characterization of basic pharmacokinetics properties to
surrogate markers, population PK and PD studies, and assessment of in-vivo
drug interactions. Three chapters in this part discuss special populations like
vii
viii Preface
disease state for example (renal and hepatic impairment), gender, race, age
(elderly and pediatric), pregnancy, and lactation. The last chapter in Part III
discusses clinical pharmacology issues related to several specific drug
classes.
Clinical pharmacology is followed by a part on biopharmaceutics. This
part starts off with a chapter on bioavailability and bioequivalence (BA/BE)
assessments for new and generic drugs followed by chapters on oral
extended release products, and when and how one can obtain a waiver for
conducting in-vivo BE studies. The last chapter in this part describes the
assessment of BE of drugs administered via routes other than oral.
There are certain situations in drug development which require
additional consideration. For example, the development of a chiral drug,
liposomal drug product, or drugs to treat situations/illnesses created by
biological and nerve poisoning agents. The last part of this book discusses
such contemporary or special topics. The last chapter in this book is a
tutorial in conducting statistical analysis of BE studies.
The FDA and other regulatory agencies continue to release guidances on
contemporary topics. For example, when this book went in to print,
guidances on pharmacogenomics/pharmacogenetics and assessment of QTc
prolongation by drugs were still being developed. This book is by no means
exhaustive and the reader is encouraged to refer to the regulatory agency
websites on these ever-evolving contemporary topics.
The chapters in this book are the result of expertise and time devoted by
many experts from the FDA and other regulatory agencies. In addition to
the scientific principles, the authors were encouraged to include key points
from the latest regulatory guidances. Further, authors have attempted to
include the regulatory requirements from other (European, Canada)
agencies and also incorporate ICH (International Conference on
Harmonization) requirements. There are 25 chapters written by 40 authors
in this book. I have made every attempt to use the same format and
terminology and avoid duplication of information. However, since this
book is aimed to be used as a teaching tool, some duplicated information
was deliberately left untouched for the sake of completeness of a chapter.
This book is intended to serve as an introductory reference text to the
pharmaceutical scientist, student, and researcher involved in the new drug
development. This book is not intended to be used as a template, but gives
the reader basic understanding of the drug development process for a new
chemical being developed as a drug.

Preface ix
Acknowledgements
I am very grateful to all the authors for generously contributing and sharing
their time, knowledge, and experience in writing this book. I am sincerely
and deeply grateful to Dr. Larry Lesko for encouraging me to work on this
idea and for his consistent support during this project. With many thanks
and gratitude I recognize my teachers, colleagues, and co-workers, from
whom I have learned a great deal.
I am thankful to Sandra Beberman, of Marcel Dekker, for encouraging
me to develop my initial idea and for her patience, optimism, and
understanding during the preparation of manuscript. I highly appreciate
Paige Force, production editor, and other copyeditors and designers, for
their careful scrutiny and invaluable support dealing with the idiosyncrasies
and language variation used by several authors.
Chandrahas Sahajwalla

Contents
Foreword v
Preface vii
Contributors xv
Part I History and Basic Principles
1. Introduction to Drug Development and Regulatory Decision-

Making 1
Lawrence J.Lesko and Chandrahas Sahajwalla
2. Evolution of Drug Development and its Regulatory Process 13

Henry J.Malinowski and Agnes M.Westelinck
3. Regulatory Bases for Clinical Pharmacology and

Biopharmaceutics Information in a New Drug Application 35
Mehul Mehta and John Hunt
4. New Drug Application Content and Review Process for

Clinical Pharmacology and Biopharmaceutics 71
Chandrahas Sahajwalla, Veeneta Tandon, and Vanitha J.Sekar
xi
xii Contents
Part II In Vitro/Pre-Clinical
5. In-vitro Drug Metabolism Studies During Development of

New Drugs 87
Anthony Y.H.Lu and Shiew-Mei Huang
6. Drug Transporters 111

Xiaoxiong Wei and Jashvant D.Unadkat
7. Principles, Issues, and Applications of Interspecies Scaling 137

Iftekhar Mahmood
Part III Clinical Pharmacology
8. Analytical Method Validation 165

Brian P.Booth and W.Craig Simon
9. Studies of the Basic Pharmacokinetic Properties of a Drug:

A Regulatory Perspective 187
Maria Sunzel
10. Surrogate Markers in Drug Development 213

Jürgen Venitz
11. Population Pharmacokinetic and Pharmacodynamic Analysis 229

Jogarao V.S.Gobburu
12. Scientific and Regulatory Considerations for Studies

in Special Population 245
Chandranas Sahajwalla
13. Conducting Clinical Pharmacology Studies in Pregnant

and Lactating Women 267
Kathleen Uhl
14. Scientific, Mechanistic, and Regulatory Issues with

Pharmacokinetic Drug-Drug Interactions 297
Patrick J.Marroum, Hilde Spahn-Langguth, and
Peter Langguth
15. Assessing the Effect of Disease State on the Pharmacokinetics

of the Drug 345
Marie Gårdmark, Monica Edholm, Eva Gil-Berglund,
Carin Bergquist, and Tomas Salmonson

Contents xiii
16. Clinical Pharmacology Issues Related to Specific Drug

Classes During Drug Development 373
Kellie Schoolar Reynolds, Vanitha J.Sekar, and Suresh
Doddapaneni
Part IV Biopharmaceutics
17. Issues in Bioequivalence and Development of Generic

Drug Products 399
Barbara M.Davit and Dale P.Conner
18. Regulatory Considerations for Oral Extended Release

Dosage Forms and in vitro (Dissolution)/in vivo (Bioavailability)
Correlations 417
Ramana S.Uppoor and Patrick J.Marroum
19. In vivo Bioavailability/Bioequivalence Waivers 449

Patrick J.Marroum, Ramana S.Uppoor, and Mehul U.Mehta
20. Bioavailability and Bioequivalence Issues for Drugs

Administered via Different Routes of Administration;
Inhalation/Nasal Products; Dermatological Products,
Suppositories 475
Edward D.Bashaw
Part V Contemporary and Special Interest Topics
21. Scientific and Regulatory Issues in Development of Chiral

Drugs 503
Chandrahas Sahajwalla, Jyoti Chawla, and Indra K.Reddy
22. A Regulatory View of Liposomal Drug Product

Characterization 525
Kofi Kami and Brian P.Booth
23. Challenges in Drug Development: Biological Agents of

Intentional Use 535
Andrea Meyerhoff
24. The Regulation of Antidotes for Nerve Agent Poisoning 543

Russell Katz and Barry Rosloff

xiv Contents
25. Bioequivalence Assessment: Approaches, Designs, and

Statistical Considerations 561
Rabindra N.Patnaik

Contributors
Edward D.Bashaw Division of Pharmaceutical Evaluation III, Office of

Clinical Pharmacology and Biopharmaceutics, Center for Drug Evaluation
and Research, Food and Drug Administration, Rockville, Maryland, U.S.A.
Eva Gil Berglund Medical Products Agency, Uppsala, Sweden
Carin Bergquist Medical Products Agency, Uppsala, Sweden
Brian P.Booth Division of Pharmaceutical Evaluation I, Office of Clinical

Pharmacology and Biopharmaceutics, Center for Drug Evaluation and
Research, Food and Drug Administration, Rockville, Maryland, U.S.A.
Jyoti Chawla University of Washington, Seattle, Washington, U.S.A.
Dale P.Conner Division of Bioequivalence, Office of Generic Drugs, Office

of Pharmaceutical Science, Center for Drug Evaluation and Research, Food
and Drug Administration, Rockville, Maryland, U.S.A.
Barbara M.Davit Division of Bioequivalence, Office of Pharmaceutical

Science, Office of Generic Drugs, Center for Drug Evaluation and Research,
Food and Drug Administration, Rockville, Maryland, U.S.A.
xv
xvi Contributors
Suresh Doddapaneni Division of Pharmaceutical Evaluation II, Office of

Monica Edholm Medical Products Agency, Uppsala, Sweden
Marie Gårdmark Medical Products Agency, Uppsala, Sweden
Jogarao V.S.Gobburu Division of Pharmaceutical Evaluation I, Office of

Shiew-Mei Huang Office of Clinical Pharmacology and Biopharmaceutics,

Center for Drug Evaluation and Research, Food and Drug Administration,
John Hunt Division of Pharmaceutical Evaluation II, Office of Clinical

Russell Katz Division of Neuropharmacology Drug Products, Office of

Drug Evaluation I, Center for Drug Evaluation and Research, Food and
Drug Administration, Rockville, Maryland, U.S.A.
Kofi Kumi Division of Pharmaceutical Evaluation I, Office of Clinical

Peter Langguth Johannes Gutenberg-University, Germany
Lawrence J.Lesko Office of Clinical Pharmacology and Biopharmaceutics,

Center for Drug Evaluation and Research, Food and Drug Administration,
Anthony Y.H.Lu Rutgers University, Piscataway, New Jersey, U.S.A.
Iftekhar Mahmood Center for Biologies Evaluation and Research,


Contributors xvii
Henry J.Malinowski Division of Pharmaceutical Evaluation II, Office of

and Research, Food and Drug Administration, Rockville, Maryland,
U.S.A.
Patrick J.Marroum Division of Pharmaceutical Evaluation I, Office of

Mehul U.Mehta Division of Pharmaceutical Evaluation I, Office of Clinical

Andrea Meyerhoff* Department of Health and Human Services, Food and

Rabindra N.Patnaik† Center for Drug Evaluation and Research, Food and
Indra K.Reddy University of Arkansas for Medical Sciences; Little Rock,

Arkansas, U.S.A.
Kellie Schoolar Reynolds‡ Division of Pharmaceutical Evaluation III, Office

of Clinical Pharmacology and Biopharmaceutics, Center for Drug
Evaluation and Research, Food and Drug Administration, Rockville,
Maryland, U.S.A.
Barry Rosloff Division of Neuropharmacological Drug Products, Office of

Drug Evaluation I, Center for Drug Evaluation and Research, Food and
Chandrahas Sahajwalla Division of Pharmaceutical Evaluation I, Office of

* Current affiliation: Clinical Associate Professor of Medicine, Division of Infectious Diseases,

Georgetown University, Washington, D.C., U.S.A.
† Current affiliation: Executive Director, Biopharmaceutics, Watson Laboratories, Inc., Corona,
California, U.S.A.
‡ Current affiliation: Global Biopharmaceutics, Drug Metabolism and Pharmacokinetics,
Aventis Pharmaceuticals, Bridgewater, New Jersey, U.S.A.

xviii Contributors
Tomas Salmonson Medical Products Agency, Uppsala, Sweden
Vanitha J.Sekar* Division of Pharmaceutical Evaluation I, Office of Clinical

W.Craig Simon Therapeutic Products Directorate, Health Canada, Ottawa,

Ontario, Canada
Hilde Spahn-Langguth Martin-Luther-University, Halle-Wittenberg,

Wolfgang-Langenbeck-Str., Germany
Maria Sunzel† Division of Pharmaceutical Evaluation I, Office of Clinical

Veeneta Tandon Division of Pharmaceutical Evaluation I, Office of Clinical

Kathleen Uhl Office of New Drugs, Center for Drug Evaluation and
Jashvant D.Unadkat Department of Pharamceutics, University of

Washington, Seattle, Washington, U.S.A.
Ramana S.Uppoor Division of Pharmaceutical Evaluation I, Office of

Jürgen Venitz Department of Pharmaceutics, School of Pharmacy, Virginia

Commonwealth University, Richmond, Virginia, U.S.A.
* Current affiliation: Aventis Pharmaceuticals, Bridgewater, New Jersey, U.S.A.

† Current affiliation: Director, Clinical Pharmacology, Experimental Medicine, AstraZeneca LP,
Wilmington, Delaware, U.S.A.

Contributors xix
Xiaoxiong Wei Division of Pharmaceutical Evaluation II, Office of Clinical

Agnes M.Westelinck* Division of Pharmaceutical Evaluation II, Office of

Clinical Pharmacology and Biopharmaceutics, Food and Drug Adminis-
tration, Rockville, Maryland, U.S.A.
* Current affiliation: Barrier Therapeutics, Princeton, New Jersey, U.S.A.

New Drug
Development

1
Introduction to Drug Development and
Regulatory Decision-Making
Lawrence J.Lesko and

The science of contemporary drug development is a tremendously complex

and costly process but it has successfully advanced our understanding of
modern diseases and has improved public health significantly by providing
society with many valuable drug treatments. A crucial step in the drug
development process is the submission of nonclinical and clinical data and
information in a New Drug Application (NDA) to the Food and Drug
Administration (FDA) by a sponsor seeking marketing authorization. A
typical new molecular entity (NME) that is the subject of a NDA has most
likely been studied preclinically for 5–7 years and has been in clinical trials for
6–7 years. The average cost of bringing an NME to market is somewhere
between 500 and 800 million dollars including the costs of lost opportunities
and lead-compound failures [1]. With this investment of time and money,
many scientists involved in drug development have explored various ways to
make drug development as efficient, and yet informative, as possible [2].
1
2 Lesko and Sahajwalla
Despite its successes, the drug development process, including regulatory

decision-making based on benefit/risk assessments, can be improved in three
areas.
1. Provide a greater understanding of human health and the

causes of diseases at a genomic or molecular level. This would
address the well-known heterogeneity of disease states that
underlies the wide interindividual variation in efficacy
observed with many common treatments. For example,
incomplete or absence of response occurs in 30–50% of eligible
patients with hypercholesteremia who are treated with
“statins.” With greater insights into health and disease,
sponsors would be more likely to identify a target protein or
receptor and to find the best NME to provide preventive,
curative, or palliative treatment for patients.
2. Improve the safety of medicines. Adverse drug reactions (ADRs)
have had a major impact on morbidity, mortality, and health
economics. In studies going back to 1974, up to the present time,
approximately 15–20% of hospitalized children and 25–30% of
hospitalized adults have experienced drug-related adverse events
[3, 4]. The overall incidence of drug-induced adverse events in
nonhospitalized patients is thought to be around 7% [5]. The
economic cost of drug-related morbidity and mortality to society
has been estimated to be almost 200 billion dollars [6]. While
there are many reasons, some of them unknown, for the
relatively high incidence of ADRs (e.g., medication errors, drug
interactions), it is thought that the majority of the risks
associated with drug therapy are known and most drug-related
adverse events are preventable [7].
3. Optimize drug doses and dosing schedules. Approximately 70%
of drug-related adverse events are due to extended
pharmacological actions. Thus, there is growing evidence to
suggest that drug doses approved for marketing may be higher
than is necessary and may be contributing to the high frequency
of serious drug side effects. A recent study that examined the
doses of 354 prescription drugs recommended in the label and
released between 1980 and 1999 found that approximately 17%
of these drugs had a reduction in dose or a new restriction for use
in special populations such as patients with renal or hepatic
disease [8]. Furthermore, it has been reported that prescribers in
their practice frequently use doses which are lower than the
FDA-approved label dose [9]. In an informal survey, it was also
found that doses approved in other countries, e.g., Japan, are

Drug Development and Regulatory Decision-Making 3
lower than those approved in the United States and most often
there are no apparent scientific rationale for these differences.
These three areas of improvement should be viewed as a challenge to the

scientific community in industry, academia, and the regulatory agencies to
engage in dialogue and scientific collaboration to optimize the drug
development process. This is especially important in light of the emergence
of new genetic technologies and our understanding of the human genome
that provides us new ways to ask important questions during the drug
development process. Indeed, the promise of personalized or predictive
medicine that stems from pharmacogenetics and pharmacogenomics means
that the benefit/risk ratio of drugs is systematically optimized by identifying
and selecting the right drug target, developing the right drug, and delivering
the right dose to the right patient.
ROLE OF CLINICAL PHARMACOLOGY
At the core of the drug development process is a fundamental understanding

of the clinical pharmacology of the drug substance. Clinical pharmacology
can be thought of as a translational science in which basic information
about the relationship between a drug’s dose, local or systemic exposure and
response (related to either efficacy or safety) is applied in the context of
patient care. Knowledge of this relationship, which is a key to successful
therapeutics, and how it is altered by the intrinsic (age, gender, renal
function, etc.) and extrinsic (diet, drugs, life-style) factors of an individual
patient is one of the major contributions of clinical pharmacology to drug
development and regulatory decision-making.
Once a lead compound with the intended pharmacological action is
identified, the step-wise process to characterize and potentially optimize
its pharmacokinetic (PK) properties (i.e., absorption, distribution,
metabolism, and excretion), as well as to minimize its pharmacokinetic
limitations (e.g., poor absorption), begins in humans as part of phase I
human clinical trials. Soon after, other principles of clinical pharmacology
[e.g., pharmacokinetic-pharmacodynamic (PD) relationships] become
critical to the evaluation and selection of the most appropriate dosing
regimen of the drug in a carefully selected target population enrolled in
phase II clinical trials. These trials form the scientific rationale for
subsequent dose selection in large-scale phase III clinical trials where the
primary goal is to provide adequate evidence of efficacy and relative safety
of the drug. Phase III trials are the most expensive and time-consuming
component of the overall drug development process and many believe that
paying careful attention to doing clinical pharmacology “homework” has

the greatest potential to reduce the failure rate of new drugs at this near-
final stage of development.
Often, in parallel with phase III clinical trials, a group of clinical
pharmacology studies, such as those in special populations, are conducted
in human volunteers to develop a knowledge database of factors influencing
drug exposure. These data are crucial for an understanding of when, and
how much, to adjust dosage regimens. Because these studies typically focus
on changes in systemic exposure, as a surrogate marker for either efficacy or
toxicity, the availability and the intelligent use of exposure (e.g., dose, PK
measurements)-response (e.g., biomarkers, surrogate clinical endpoints,
clinical outcomes, PD) relationships to interpret the results of these studies
become critical to information for various sections of the product label.
These studies can be broadly classified into two broad categories: (1) those
dealing with patient-intrinsic factors that include gender, age, race, diseases
states (primarily renal and/or hepatic impairment), and genetic (e.g., activity
of cytochrome P450 enzymes) factors, and (2) those dealing with patient-
extrinsic factors that include drug-, herbal- and nutrient-drug interactions,
environmental variables (e.g., smoking, diet), and lifestyle factors.
ROLE OF BIOPHARMACEUTICS
Related to the science of clinical pharmacology, biopharmaceutics can be

thought of as the body of scientific principles applied to convert a well-
characterized drug substance to an appropriate, and potentially optimized,
drug product. At the heart of biopharmaceutics is a thorough understanding
of the physical, chemical and biological properties of the drug substance
related to absorption (e.g., solubility, stability and intestinal permeability)
and how to utilize these data to decide on the best route of administration
and to develop a successful dosage form. The development of an initial
formulation for a drug substance entails the study of drug product
dissolution under a variety of environmental conditions (e.g., pH), and
linking the resulting rate and extent of dissolution to the subsequent rate
and extent of absorption (i.e., bioavailability or BA). These so-called in
vitro-in vivo correlations (IVIVC) are important to early optimization of
formulation performance in order to achieve systemic plasma drug
concentration-time profiles later in human clinical trials with the greatest
chance for therapeutic success.
Not infrequently, the final, to-be-marketed formulation of the active drug
substance is different than the initial formulations used in either early or late
clinical trial phases of development. Biopharmaceutics plays a critical role in
linking the in vivo performance or BA of each of the early formulations (i.e.,
reference formulations) to the final (i.e., test formulations) formulations.

The standard study to assess comparative BA of the test and reference

formulations is the bioequivalence (BE) study. Often, the results of BE
studies are expressed as measures of exposure, such as area under the
plasma concentration-time curve (AUC) and peak or maximum plasma
concentration (Cmax). The ratio of these in vivo measurements (test/
reference) are usually statistically reported as 90% confidence intervals (CI).
BE is declared if the 90% CI is between 80 and 125% (“goalposts”).
However, if the 90% CI is either partially or completely outside these
“goalposts”, therapeutic equivalence is determined by integrating the
clinical pharmacology information about exposure-response relationships
into the regulatory decision-making process.
REGULATORY REVIEW
Within the Center for Drug Evaluation and Research (CDER) of the FDA,
the regulatory review of clinical pharmacology and biopharmaceutics
studies is the responsibility of the Office of Clinical Pharmacology and
Biopharmaceutics (OCPB). The mission of OCPB has patient care and
therapeutics as center stage, and this is reflected by the scientific goals of
clinical pharmacology and biopharmaceutics, that is, to critically study,
thoroughly understand, and successfully identify (1) the right dose, in (2)
the right dosage form, for (3) the right patient. The final step is to
responsibly translate this knowledge to the product label with appropriate
information about the use of the drug/drug product in the clinical
pharmacology, precautions, warnings, contraindications, and/or dosage
and administration sections of the package insert. This is indeed a critical
step in the review process, since labeling a drug for use in the manner that is
intended for patients to use it (or not use it) is one of the most important
ways of risk management for ADRs.
OCPB’s review process is based on a paradigm known as the Question-
Based Review, or QBR [10]. It recognizes that it would be unreasonable to
expect that everything will be known about the clinical pharmacology (CP)
and biopharmaceutics (BP) of a drug/drug product at the time of NDA
submission. Accordingly, the QBR emphasizes the importance of the
reviewer’s responsibility to ask the right questions related to the efficacy and
safety of new medicines based on the clinical pharmacology and
biopharmaceutics database provided by the sponsor in a NDA, and also to
identify what is important but not known about the drug. The latter may be
the basis for postmarketing studies (phase IV commitments). There are
many critical principles in applying the QBR but two stick out the most
when reviewing CP and BP studies: (1) analyzing study results and
integrating knowledge thoughtfully across studies, and not just reviewing

studies in isolation from one another, or necessarily in the chronological

order in which they were conducted, and (2) interpreting results of CP and
BP studies in the overall context of what is also known from the nonclinical
chemistry, pharmacology and toxicology data, and the clinical efficacy and
safety information, and not just to focus on providing a narrow-focused CP/
BP report to medical officers. To meet these responsibilities, reviewers are
strongly encouraged to act credibly and to communicate extensively with
other professionals during the review process.
VIEW TOWARD THE FUTURE
Clinical pharmacologists and biopharmaceutical scientists have an

opportunity, as much as any professional, to lead the pharmaceutical
industry and regulatory agencies in leveraging their science and technology
for achieving future breakthroughs in therapeutics. The process of marrying
comprehensive biopharmaceutical information to clinical pharmacology
data, and integrating that knowledge into what is known about drug
efficacy and safety, will bring the drug development enterprise a step closer
to realizing the dream of individualized medicine. Part of this process will be
leveraging several existing fundamental technologies and new scientific
discoveries to a greater extent.
Pharmacogenetics (PGt) and Pharmacogenomics (PGx)

While no consensus on definitions is at hand, for the purpose of this chapter
PGt can be thought of as the study of the genetic variability in PK among
individuals, affecting liver enzymes that metabolize drugs and transporters
that determine BA and drug distribution. PGx, closely related to PGt, may
be defined as the study of genetic variability, including that of drug receptors
(PD), among individuals, affecting the rest of the genome that regulates drug
response. Many believe that PGt and PGx are at the core of future drug
development processes with applications ranging from new knowledge
about the molecular basis of diseases to identification of new genes or gene
products (e.g., protein) that serve as novel drug targets. There are several
significant industry examples of the impact of PGt and PGx. These include
(1) the comarketing of trastuzumab (Herceptin, Genentech) and a
diagnostic test (HercepTest) for patients with breast cancer whose tumors
have overexpressed HER 2 activity [11], (2) a gene-based diagnostic marker
that has the potential to identify at-risk patients with HIV for
hypersensitivity to abacavir (Ziagen, GSK), (3) haplotypes that have the
potential to be used as diagnostic tests to optimize the selection of approved
HMG Co-A reductase inhibitors (“statins”) in patients with

hypercholesteremia, and (4) potential genetic markers to identify patients

with rheumatoid arthritis who are responders to IL-1 and TNF-inhibitors. A
regulatory perspective on PGt and PGx has recently been published and
regulatory agencies worldwide generally are optimistic that these sciences
will, in time, profoundly transform the drug development and regulatory
review processes [12].
However, closer attention needs to be paid to what is already known
about PGt with an eye toward how this information can be integrated into
current standards of patient care to reduce the incidence of ADRs. For
example, it has been reported that of the top 27 drugs frequently cited in
ADR reports, 59% are metabolized by at least one enzyme having poor
metabolizer (PM) genotype. Eleven of the 27 drugs (38%), mainly used for
cardiovascular and CNS diseases, are metabolized specifically by cytochrome
P450 (CYP) 2D6 [13]. Despite the strong suggestion that knowing a patient’s
CYP 2D6 genotype (or phenotype), and adjusting doses downwards or
upwards depending on the genotype, would positively influence benefit/risk
of therapy, CYP 2D6 genotyping is not recommended in any package insert
of approved products. There are a variety of reasons for this, but as genotyping
tests for CYP enzyme activity become more widely available and cost-effective,
clinical pharmacologists will have the responsibility to ask the right questions
about genetic polymorphism and to act responsibly on the information during
drug development and regulatory review.
In the broad world of PGx, there will be greater reliance on global DNA
sequencing and candidate gene studies to discover genes and genetic
biomarkers that play a role in assessing disease progression and variability
in drug response. Clinical pharmacologists will have opportunities to
explore associations between gene variants, in the form of single nucleotide
polymorphisms (SNPs) or combinations of SNPs (haplotypes), to better
understand variability in drug response and dosage requirements. In
addition, complementary PGx technologies, such as gene-chip microarrays
and quantitative polymerase chain reaction (PCR), will provide additional
insights into the genetic basis of disease and drug response which will
impact clinical therapeutics in terms of measuring disease- and drug-
induced differences in expression profiles and providing multiple biomarker
panels to associate with drug therapy.
Assay Development
It is well known that chemical assays of high quality (i.e., adequate
sensitivity, selectivity, and reproducibility) are essential to obtaining credible
data in clinical pharmacology studies (e.g., PK) and biopharmaceutics
studies (e.g., BE). However, in the future, assay development that includes
more sophisticated technologies and more attention to detail will be needed.

For example, there are many pharmacological or physiological biomarkers

of drug activity which are used in analyzing exposure-response relationships
for the purpose of making decisions in drug development or regulatory
review, where evidence of validation of the measurement of the response
component is incomplete or missing. In addition, with the evolution of PGt
and PGx, principles of validation of new technologies such as mass
spectrometry (proteomics), high-throughput DNA sequencing, and
expression profiling (microarrays) will need to be established to ensure
credible interpretation and use of these data. Each of these newer
technologies, in contrast to traditional technologies, will provide a
tremendous amount of information about changes in gene expression and
potentially useful biomarker panels. The bioinformatics software used to
mine these data sets is not standardized at the moment, and as a result
various association algorithms, cluster analyses, and SNP and haplotype
identification methods are used from company to company. The potential
for interlaboratory differences in interpretation is enormous and consensus
on how to use these tools reliably will be important in clinical pharmacology
and biopharmaceutics studies of the future.
Modeling and Clinical Trial Simulation (CTS)

Development and validation of models for exposure-response datasets have
been widely used by clinical pharmacologists during drug development and
regulatory review to understand the nature of dose-response and PK-PD
relationships and to predict alternative clinical scenarios. There are many
examples of the value of modeling in terms of improving drug development
and regulatory review [14]. In the future, modeling of biological systems at
the cellular level, disease progression models, and models for quantitative
assessment of risk will take on greater importance in CP studies. More
recently, CTS or computer assisted trial design (CATD) methodologies have
been advanced as tools to use phase I and phase II exposure-response
information to design phase III trials, predict trial outcomes in terms of
efficacy and safety, and allow for more informed decisions on benefit/risk
analysis and the economics of drug development programs [15]. CATD,
while not routinely used in drug development and regulatory review, is likely
to take on more importance as our understanding of the causes of disease,
disease progression, molecular drug targets, and drug pharmacology/
toxicology increases through the co-evolution of genetics and genomics.
Diagnostic Tests and Kits

As PGt and PGx mature, it is highly likely that gene-based diagnostic tests
and kits using genetic markers will significantly influence drug

development and regulatory review. These tests and kits will not only be
used on patient blood or tissue samples to diagnose diseases when they are
present, but will also be able to (1) predict the probability of developing-
diseases in the future, (2) identify patients who are most likely to be
responders or nonresponders, (3) select the most appropriate dose for a
given individual, and (4) select the best drug in a class once a decision is
made to institute drug therapy. To date, there are relatively few diagnostic
test kits approved by FDA, although in the future this would be desirable.
HercepTest (Dako Corporation) and PathVysion Her-2 DNA FISH (Vysis)
have been approved by FDA to measure HER 2 activity prior to making a
medical decision to administer Herceptin to women in advanced stages of
breast cancer, and HIV-1 TruGene Assay (Applied Sciences/Visible
Genetics) has been approved to measure HIV resistance and to provide
drug treatment options for patients with AIDS. FDA approval of gene-
based diagnostics would provide many advantages such as assuring high
quality reagents, validated reference standards, standardized assay
procedures and protocols, and greater acceptance of these tests by patients
and physicians. Interpreting the test results for physicians, by bridging this
information to package inserts, is likely to become an important
responsibility of clinical pharmacologists in the future.
Knowledge Management (KM)
For the purposes of this chapter, KM is defined as the marriage of science,

bioinformatics, and computer technology to more effectively assess and
utilize the ever increasing amounts of clinical pharmacology and
biopharmaceutics data arising from drug development. As an example,
modern NDAs may contain more than 60 CP and BP studies, and each
study contains many more pieces of data than ever before. In order to
conduct a meaningful and thorough analyses of these data and to learn as
much as possible about the drug/drug product, industry and regulatory
scientists will need the capability that computer visualization and analysis
software can offer. Applying web-based data management will enable
endusers to (1) use information across studies better, (2) make more
efficient and informed decisions about benefit/risk, and (3) create learning
databases that can be effectively queried to compare CP and BP attributes
across drugs and therapeutic areas. Visualization software is also a
powerful way to communicate important CP and BP information to those
in other disciplines in order to make maximum use of the scientific data at
hand.

SUMMARY
The current mission and goals of clinical pharmacology and

biopharmaceutics is highly likely to expand and be transformed in the future
as the new tools, technologies, and expectations (as described above and in
the following chapters) become reality. Many of the questions about
efficacy, safety, benefit/risk, drug dosing, and drug product performance
will be tailor made for the scientists in CP and BP. These scientists will have
to integrate their knowledge with other disciplines more broadly to take a
leading role in drug development and regulatory decision-making. The
efforts of clinical pharmacologists and biopharmaceuticists, if future
challenges are accepted by the profession, will have the potential to
introduce innovation and ultimately impact the standards of medical care.
How CP and BP data is interpreted and applied in the future will affect risk
assessment, risk management plans, and drug development and regulatory
decisions. The quality of CP information in drug product labels and the
setting of standards and specifications based on BP data to assure consistent
drug product performance over time in the marketplace will likely impact
the effectiveness and, perhaps most importantly, the safety of new
medicines. This is, without a doubt, a common and meritorious goal shared
by clinical pharmacologists and biopharmaceuticists whether they practise
in industry or in regulatory agencies.
REFERENCES
1. Tufts Center for the Study of Drug Development: Outlook 2002; http://
csdd.tufts.edu/InfoServices/OutlookPDFs/Outlook2002.pdf.
2. Lesko, L.J.; Rowland, M.; Peck, C.C.; Blaschke, T.F. Optimizing the Science of
Drug Development—Opportunities for Better Candidate Selection and
Accelerated Evaluation in Humans. J Clin Pharmacol 2000, 40 803–814.
3. Miller, R.R. Hospital Admissions Due to Adverse Drug Reactions—A Report
from the Boston Collaborative Drug Surveillance Program. Arch Intern Med
1974, 134, 219–223.
4. Mitchell, A.A.; Goldman, P.; Shapiro, S.; Slone, D. Drug Utilization and Reported
Adverse Reactions in Hospitalized Children. Am J Epidemiol 1979, 110, 196–
204.
5. Lazarou, J.; Pomeranz, B.H.; Corey, P.N. Incidence of Adverse Drug Reactions
in Hospitalized Patients—A Meta-Analysis of Prospective Studies. JAMA 1998,
279, 1200–1205.
6. Ernst, F.R.; Grizzle, A.J. Drug-Related Morbidity and Mortality: Updating the
Cost-of-illness Model. J Am Pharm Assoc 2001, 41, 192–199.
7. Kohn, L.T.; Corrigan, J.M.; Donaldson, M.S., Eds. To Err is Human: Building a
Safer Health System, Institute of Medicine, The National Academies Press, 2000.

8. Cross, J.; Lee, H.; Westelinck, A.; Nelson, J.; Grudzinskas, C.; Peck, C.
Postmarketing Drug Dosage Changes of 499 FDA-Approved New Molecular
Entities, 1980–1999. Pharmacoepidemiology and Drug Safety 2002, 11, 439–
446.
9. Cohen, J.S. Overdose: The Case Against the Drug Companies—Prescription
Drugs, Side Effect, and Your Health, Penguin Putnam, Inc., 2001.
10. Lesko, I.J.; Williams, R.L. The Question-Based Review: A Conceptual Framework
for Good Review Practices. Applied Clinical Practice 1999, 8, 56–62.
11. Dako, A.S. Cytomation, Inc. http://www.dakousa.com.
12. Lesko, L.J.; Woodcock, J. Pharmacogenomic-Guided Drug Development—
Regulatory Perspective. The Pharmacogenomics Journal 2002, 2, 20–24.
13. Philips, K.A.; Veenstra, D.L.; Oren, E.; Lee, J.K.; Sadee, W. Potential Role of
Pharmacogenomics in Reducing Adverse Drug Reactions—A Systematic Review.
JAMA 2001, 2867, 2270–2279.
14. Derendorf, H.; Lesko, L.J.; Chaikin, P.; Colburn, W.; Lee, P.; Miller, R et al.
Pharmacokinetic/Pharmacodynamic Modeling in Drug Research and Devel-
opment. J Clin Pharmacol 2000, 40, 1399–1418.
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to Improve Decision-Making in Clinical Drug Development. Eur J Drug Metab
Pharmacokinet 2000, 25, 49–58.

2
Evolution of Drug Development and its
Regulatory Process
Henry J.Malinowski and

Agnes M.Westelinck*
The history of clinical pharmacology over the past 100 years may be thought
of as a gradual progression from the use of potions and other sometimes
dubious concoctions to the complex drug development process seen today
[1]. The future of clinical pharmacology has been described as academia,
industry, and government working together to advance science, develop new
drugs, and improve the quality of life of mankind [2]. Efforts such as the
International Conference on Harmonization (ICH) have promoted unification
of regulatory policies, including those related to clinical pharmacology. More
than 35 harmonized ICH Guidelines are available [3] and the recently
harmonized Common Technical Document provides for a common format
for new drug and biological regulatory submissions. Following are
perspectives from Europe and the United States on the progress of clinical
pharmacology over the years, in these two major regions of the world.
* Current affiliation: Barrier Therapeutics, Princeton, New Jersey, U.S.A.
13
14 Malinowski and Westelinck
DRUG DEVELOPMENT IN EUROPE
Early Days
Clinical pharmacology, the science of drug actions in humans, started its
development in the 19th century. Test animals were increasingly used in
pharmacology research. In France, Francois Magendie (1783–1855) played
a prominent role. He is known to many for his description of the foramen of
Magendie in the brain but could be thought of also as one of the most
important founders of modern pharmacology. Czech Jan Evangelista
Purkinje (1787–1869), whose name is linked to large nerve cells in the brain
(Purkinje cells) and to conducting tissue in the heart (Purkinje fibers), was
one of the first to study drugs in healthy subjects, an unusual step, to avoid
interference by illnesses when studying drug characteristics [4]. In 1805,
German pharmacist Friedrich Serturner isolated the pure active ingredient
in opium. He named this chemical morphine, after Morpheus, the Greek
god of dreams. Serturner’s discovery was the first isolation of an active
ingredient. For many years he experimented on himself and others to
explore the effects of the alkaloid.
In the 17th century, a controlled study design was described. Jan Baptista
van Hellemont (1578–1644), a physician in Brussels, had proposed to his
opponents to settle a dispute about wound treatments. Several hundred
patients were to participate in an experiment, with vitriol or bloodletting
treatments assigned by lottery to each individual patient. Results were to be
judged by “the number of funerals” on each side. It is only in the 20th
century that the randomized controlled study design became generally
accepted. The double blind randomized study conducted in the late 1940s
by the British Medical Research Council confirming the effect of
streptomycin on tuberculosis was to become a classical example. With the
emergence of the chemical industry in the second half of the 19th century,
drug manufacturing by chemical synthesis became possible and a number of
pharmaceutical companies emerged.
Several drugs to treat serious diseases were discovered. Due to
insufficient pharmacological knowledge those drugs were probably too
easily introduced. The American government realized an important role to
play. Legislation in 1938 and later in 1962 required manufacturers to show
respectively safety and efficacy of drugs. The American example was
followed in Europe with some delay. In the Netherlands the first such
legislation was introduced in 1958. But it was only after the thalidomide
tragedy in the 1960s that an official agency to evaluate drugs started to
operate efficiently in this country. Similarly, in the United Kingdom it was
not until the Medicines Act was introduced in 1972 that evidence of efficacy
as well as safety was required as a condition for granting a product license.

Drug Development and its Regulatory Process 15
The legal obligation to demonstrate safety and efficacy before market

introduction stimulated the development of clinical pharmacology as a new
scientific discipline. The development of clinical pharmacology is a logical
consequence of the pharmaceutical revolution in the beginning of the 20th
century and the increasing contribution that drug treatments have made to
medical practice in the second half of the century [4, 5].
Clinical Pharmacology
Clinical pharmacology, the science of interactions between men and drugs,

was forged as an established medical discipline in the late 1950s and early
1960s in the United States, the United Kingdom, and Scandinavia. By 1970,
it had been recognized by World Health Organization (WHO) and in the
same year the Clinical Pharmacology section of the British Pharmacological
Society was formed. In 1974 the British Journal of Clinical Pharmacology
was launched. Clinical pharmacology has developed unevenly within the
European region and indeed throughout the world. It has developed rather
at a faster pace in some countries (e.g., the United Kingdom, Scandinavia)
but slower in others. The functions of clinical pharmacology were defined
30 years ago in a WHO report as research, teaching and service functions to
enhance the “scientific study of drugs.” Pharmacological service functions
are referred to functions aiming to solve problems in drug therapy, not to
traditional clinical work. In retrospect it is felt in Europe that most clinical
pharmacology groups who lived up to the recommendation of this WHO
report have evolved favorably, while many of those who did not, have
disappeared [6].
There are different descriptions of clinical pharmacology. It is considered
as both a research discipline (interdisciplinary) and a clinical specialty
(specified training of MDs). Under ideal circumstances they work closely
together, and there is a career ladder for both. At times, there has been
tension between a conservative clinical specialist approach, at the cost of
isolation, and a broader multidisciplinary-in-touch approach. However, to
meet various challenges in Europe, old barriers divided along traditional
subject lines, are being replaced in both academia and industry by
interdisciplinary teams [6].
Four decades of clinical pharmacology research (1960–2000) have
emphasized different aspects of the discipline (see Table 1) from controlled
clinical trials and drug metabolism during the early 1960s to molecular
pharmacogenetics and pharmacoeconomy during the late 1990s [6] (also
see Section 2 of this chapter).
In Europe, clinical pharmacology continues to be driven by a thriving
pharmaceutical industry, much of which is West-European based. Its

TABLE 1 Four Decades and Different Aspects of Clinical Pharmacology [8]
development has been underpinned by the recognition that newly available

drugs must be assessed in unbiased controlled clinical trials designed,
conducted, and analyzed to the highest possible standards. Meanwhile,
understanding of potential mechanisms of drug actions has improved,
increasing the number of target sites for new drug development. Improved
measurement techniques of both drugs and their metabolites, and the body’s
response to them, have increased the understanding of pharmacokinetics
and pharmacodynamics [7].
Evolution in Clinical Drug Development

Globalization
Drug development is undertaken today mostly in a globalized industry
where companies tap international sources of technology. European
companies nurture U.S. as well as European scientific bases and vice versa.
Traditional domestic companies are mostly less innovative and rather
persist through marketing based strategies and protection [8]. Current
trends in drug development are therefore global in nature. The items
described in this section however reflect insights and opinions from
European sources.
New Needs and Concepts

The implementation of genomic research combined with progress in
discovery techniques has significantly increased the number of potential
drug candidates for a series of diseases for which there are currently no or
only insufficient treatments. Due to the present system, many of these
candidates never reach the patient because of bottlenecks in, and limitations
to, the drug development process (see Table 2). In the early 2000s, an

TABLE 2 Bottlenecks in Traditional Drug Development [6]
apparent downturn in productivity in pharmaceutical R&D has been

observed. This is illustrated by the fact that the European Medicines
Evaluation Agency (EMEA) has willingly given back part of its approved
budget in 2002 because the anticipated number of new drug applications
had not been forthcoming. European scientists from industry, academia,
and drug regulators have been discussing the so-called “crisis.” Many share
the opinion that the rational way to reverse the trend of dwindling
productivity is to introduce new faster methodologies and modern
technology at every step of the development process [9–12].
To address new needs, a series of new concepts and techniques have been
introduced in European drug development:
The need to predict the “developability” in the selection of potential drug

candidates to go forward to full drug development. Early testing is
expected to be discriminating while predictive of potential future
problems, especially with respect to toxicity in humans [11].
The need to predict the probability of therapeutic and commercial
success. Due to increasing costs of drug development and marketing
competition, companies need an early answer to the likely clinical and
commercial success with abandonment of the compound if the target
profile is not likely to be met, ideally after the first human study [13].
In the end, economics are key considerations in drug development
[14].
The increasing use of well-established techniques of PK modeling and the
evaluation of dose-concentration-effect relationships (PK/PD) for both
desired and undesired effects.
The use of rapidly evolving computer modeling and simulation
techniques especially into difficult areas such as cancer and pediatric
studies [11].
The need to optimize the dosing regimen early in clinical development.
Traditional drug development, based on the “maximal tolerated dose”

approach or fractions thereof, has often resulted in overdosing.

However, clinical trials at too high a dose may attribute an
unacceptable safety profile to an otherwise good drug [13]. Moreover,
European regulatory authorities typically require an appropriate dose-
finding study and demonstration of both the maximal tolerated and
minimal effective dose.
Clinical development divided into two parts. “Exploratory”
development or “proof-of-concept” which may require as little as one
study and typically covers Phase I and Phase II (typically, Phase I
studies conducted in healthy volunteers and Phase II in patient
population) in the traditional theme, followed by “full” development
and completion of the registration dossier. This approach is
particularly important to innovative biotechnology companies which
are considered of great value for the future. The probability of
attracting a partner, and the value of partnership to the initial
company, will depend heavily on whether the “proof-of-principle”
point has been reached [13].
The use of well-validated surrogates which can substantially shorten
clinical development time or time to reach a critical decision point.
Biomarkers (less validated) may be useful in decision making,
although a larger amount of data is usually required to offset the
uncertainty. New biomarkers are explored in preclinical development
and link preclinical pharmacology and toxicology with the design and
interpretation of early human studies [13].
Pharmacogenetics gives researchers a powerful tool in the understanding
of how genetic variation contributes to variations in response to
medicines [15, 16]. Many individual and ethnic variations in drug
metabolism have already been shown to be due to genetically
determined variations in metabolic enzyme activity, particularly
cytochrome P450 enzyme subtype polymorphisms. European
regulators therefore require the testing of relevant drugs in target
groups of poor or extensive metabolizers [17].
Integration of Knowledge
Projected needs of the pharmaceutical industry are related to the need for
broad expertise to deal with increasingly complex projects and the
integration of specialist knowledge. Optimization of the drug development
process requires technical and scientific expertise in many areas. In some
disciplines, such as genetics (human polyphormism), mathematics
(modeling, simulation), bioinformatics (prediction), and information
technology (including pharmacometrics and information management),
there is a lack of well-trained experts. Moreover, due to the

multidisciplinary nature of drug development, knowledge covering a range

of disciplines is required [9].
An expected central challenge of the pharmaceutical industry in the
coming years is the management of complex information. Many
shortcomings in drug development can be attributed to insufficient use of
available knowledge. The interfaces between the various phases of the R&D
process have to be eliminated and a seamless discovery-development process
established, ensuring that all knowledge and data are maintained and put to
maximum use throughout (Fig. 1). New standards for handling complex
data and standardization of the format for knowledge-exchange are
required (A.Cohen, personal communication, 2001). This involves,
developing IT-supported information data management and
decisionmaking process [9]. For example, very promising new standards are
to be used in view of the International Harmonization (ICH) initiatives, the
Common Technical Document (CTD), and the Electronic Common
FIGURE 1 Integration of functions. Courtesy of A.Cohen, Center for Human Drug

Research, Leiden, The Netherlands, Phase I studies tailored towards proof-of-
concept. Personal communication, 2001.

Technical Document (e-CTD). The aim is to provide a harmonized format

and content for new product applications to be used with regulatory
authorities in different regions of the world.
New Approaches in the Real World

The initial goals of drug evaluation have been modified to include new
questions directed at goals other than drug safety and efficacy. For example,
testing a drug in a population representing the “real” world setting has
become a major basis for phase III trials and for establishing “evidence-
based” pharmacotherapy.
Other new questions that have been asked are “How should the
physician and patient be advised to use the drug?” and “Is the drug better or
similar to a drug already available?” In a sense, clinical trials have evolved
from a role in drug development to physician education and competitive
marketing [18].
A frequently forgotten aspect of drug development, which in some
respects is the most important of all, is defining the drug labeling, the
European Summary of Product Characteristics (SmPC). This document
should provide essential information for the health care professional and is
the basis for patient instructions and prescribing guidelines. This document
must be accurate but needs also to be easily understood [5].
Risk and Benefit

The standards of safety expected for an agent which may be lifesaving and
one which relieves minor symptoms should not be the same. Perceptions
on the appropriate balance of risk and benefit however vary widely,
including nationally. Based on evidence of efficacy, which may be
uncertain, together with limited safety data, licensing decisions may need
to be made on as much a judgmental as a scientific basis [5]. While formal
analysis of risk and benefit for a particular drug can be carried out,
comparative risk assessment with similar drugs is also considered useful
(see next paragraph).
Efficacy and safety have traditionally been the most important
influential bases to make decisions. In the future, priorities may also be
more influenced by costs and expected benefits of drugs on the market. At
present pharmacoeconomic data are required for requesting
reimbursement in countries such as Netherlands, United Kingdom,
Denmark, Finland, Norway, and Portugal. In the future more information
regarding the efficiency of the drug as compared to available drugs may be
needed, thus magnifying the social value of the resources invested on drug
expenditure [19].

At the end, drug development should contribute to the use of the most
appropriate drug to the right patient in an optimal dosage schedule with the
right information and at a reasonable cost.
Considerations on Study Design
During the 1990s, the importance of properly designed early trials (Phase I
and II) has led to dramatic changes in their design. These changes have
included both proper randomized, double blinded designs and increased
sample sizes. Although there are different opinions on how best to use data
from Phase II in the present process, there is little doubt concerning the high
level of information likely to be available at the end of Phase II and the
conduct of too many Phase III and IV trials may be considered redundant or
unethical [18].
There are global concerns that activities carried out during the later
stages of clinical trials are balancing on the edge of inappropriate activities.
Regulatory authorities in Europe have in a sense addressed these issues by
their request, in specific situations, for comparative trials of marketed drugs.
As the goal of these trials is often to show equivalence, they, however, tend
to be more difficult to conduct and to require larger number of patients.
Occasionally, global pharmaceutical companies have sought approval on
the basis of placebo-controlled trials in the United States and have added
active control comparative trials to register in Europe [18].
Problem Solving by the Entire Community
Mistakes in the design of a drug trial are usually reported as drug failure
rather than insufficient expertise, marketing influence, inadequate
regulatory management, or improper patient enrolment and follow up. The
assumption has been made that these are problems for the pharmaceutical
companies to solve. The regulatory role is simply to identify them and reject
the failed studies. This might be considered false. It might be considered a
problem created by the process of clinical trials, which should be solved by
the entire healthcare community [18]. To address this and to reinforce the
success of the European Agency, specific changes have been proposed to the
European Commission to enlarge the scope of the Agency’s activities beyond
the evaluation of medicinal products, by strengthening its role as a scientific
adviser.
“New Safe Medicines Faster” in Europe
Competitiveness of the Industry

Pharmaceutical companies based in Europe have traditionally played a
leading role in developing new drugs, the industry making a significant

TABLE 3 Objectives of New Safe Medicines Fast in Europe [7]
contribution to the health and economy of European Union (EU)

communities. Many of the top pharmaceutical companies reside in the EU
and Switzerland and the European pharmaceutical industry has
traditionally held a world-leading position. The trend in the late 1990s,
however, indicated that U.S. companies have perhaps taken over the
leadership role, showing the U.S. industry’s superior ability to translate new
technologies into marketable medicines [9].
However, initiatives to improve the EU competitive situation are the topic
of agendas and programs of EU professional and trade organizations and a
“New Safe Medicines Faster” initiative has been recognized for support by
the European Commission [11]. Within Europe, medicinal development
may still be hampered by barriers put up by the legislation of individual
nations, by fragmentation and by suboptimal cooperation among the
industry, academia, and authorities. The need for new revised European
standards and for pan-European interdisciplinary networks is recognized
and addressed [9].
Initiatives to Exploit Huge Opportunities

Proposed key actions are to promote basic research, new leading
technologies, and new interface research, including management of the
enormous quantity of diverse data that the development of drugs delivers.
Networking is considered essential and the creation of centralized databases
and database networks at a European level is suggested. New European
platforms for regulators and researchers are recommended to design the
necessary changes to the drug development process in partnership and bring
about improvements in capacity, efficacy, and speed (Table 3). The purpose
is to exploit the enormous opportunities created by the genomic revolution
and modern drug discovery for the generation of new medicines to the
benefit of the European citizen [9].

The European System for Approving Medicines

Coordinating Scientific Resources
The role of national regulatory authorities in Europe has changed since the
EMEA came into operation in 1995, after several years of cooperation
among national authorities at a European level. The EMEA is a technical
agency coordinating the scientific resources made available by the national
authorities to provide high quality drug evaluations, to advise on
development programs and to provide useful and clear information to the
users. In addition to their country specific responsibility, national
authorities now also investigate medicines for decisions at the EU level, in
close collaboration with the drug regulatory authorities in other European
countries [20].
To Promote Public Health and Free Circulation of Medicines
The European System offers two routes for granting authorizations. A
company can or must, depending on the type of product, seek centralized
approval, which means an authorization valid for the whole EU. The
centralized procedure is compulsory for biotechnology products and
optional for innovative conventional products. In this case the application is
dealt with administratively by the EMEA. Independent evaluations are
conducted by two selected members of the European scientific committee
(named CPMP, Committee for Proprietary Medicinal Products).
Multidisciplinary teams, coordinated by the selected members, perform
those evaluations and discuss their conclusions with the other members. The
European Commission makes final decisions after the CPMP has expressed
an opinion following its scientific debate.
For innovative conventional products a company can instead choose the
route based on mutual recognition of national decisions. The European
System affords many advantages. New medicines come to market faster,
which of course benefits patients and industry. Also, by utilizing the
collective competence of several national drug authorities, the quality and
objectivity of evaluations can be improved, duplication of work is avoided,
and harmonized opinions and labeling throughout the EU becomes
available.
An important part of this European-oriented work also revolves around
developing new standards and requirements in the face of rapid scientific
discoveries and development of new medicines. The intended end result is to
promote public health and free circulation of medicines [20].
Broad Level of Satisfaction
In 2000, an extensive consultation [21] was carried out on behalf of the
European Commission to review the operation of the new European System

since 1995. It has revealed that there is a broad level of satisfaction about
the system from ministries, patient and professional associations, regulatory
authorities, and industry, although improvements can be made and new
challenges exist.
There is a general feeling that the system has contributed to the creation
of a harmonized EU market for medicinal products and that it provides a
strong foundation for an efficient regulatory environment. There is also a
general perception that assessment of products to date has provided a high
degree of protection to the public health. This is despite the fact that there
have been withdrawals from the market of products already authorized.
This is considered consistent with increasingly effective pharmacovigilance
procedures and the bias toward products developed on the leading edge of
science.
Comparative Observations
From the same consultation in Europe, comparative observations upon the
regulatory frameworks in the EU and United States have revealed a
perception that the EU is taking a more risk-adverse approach to assessment
as compared with the FDA’s policy of risk management. Specific instances
would exist where products were removed, or threatened with removal,
from the EU market because of perceived safety concerns, while the same
products were dealt within the United States by the imposition of specific
warnings in the label [21]. Comments were made about a similar level of
conservatism in the EU in the approach to the review of products in
specialist areas such as oncology and a greater willingness to embrace new
therapies in the United States [21].
Analysis of Outcomes
An analysis of outcomes of applications in the Central Procedure from 1995
to 1999, published by the EMEA [21], has shown 72% (97/135) positive
outcomes, i.e., drug approvals. For applications with a negative outcome,
methodological concerns over study design, choice of endpoint, comparator,
and selected population were raised more frequently than over those with a
positive outcome. FDA had authorized 13 (34%) of the 38 applications that
had a negative outcome in the EU. This may be explained by a different
attitude toward data requirements e.g., requirements for controlled data, by
the availability to FDA of additional regulatory tools, e.g., conditional
approvals, and by the limited use of EMEA scientific advice (11%) prior to
submission [22].
It is expected that the Reform of EU Pharmaceutical Legislation,
proposed in 2001, will influence the regulatory environment
significantly [23].

DRUG DEVELOPMENT IN THE UNITED STATES
The modern uses of clinical pharmacology data in the United States may be
thought of as having several phases, beginning with early efforts in the
1970s, which related to the increased availability of sensitive and specific
analytical methods around that time. This was followed by application of
these capabilities to various areas such as the study of specific
subpopulations. Further implementation has emphasized the link of
pharmacokinetic data to clinical safety and efficacy data. Most recent
emphasis has included better understanding of drug interactions and
optimal dose adjustment for various sub-populations. Communication of
information and recommended approaches has been facilitated by the
preparation of FDA Guidances as well as ICH Guidelines.
Era of Pharmacokinetic Studies

The modern era of drug development related to clinical pharmacology
studies may be thought to have begun in the 1970s. A key component was
the development of bioanalytical methods needed to accurately detect
plasma concentrations of administered drugs. This aspect has continued to
improve until it is now possible to measure plasma levels for nearly every
drug under development. This is an important factor in the study of the
relationships of dose, exposure, and effect.
An important regulatory milestone was the creation of the distinct Human
Pharmacokinetics and Bioavailability Section of NDAs [24]. This established
a section in each NDA in which are contained all clinical pharmacology and
biopharmaceutics studies. Prior to what is called the NDA rewrite, NDAs
were not very consistent in content, and information to be included was not
very precisely defined or well organized. When this Format and Content
Guideline was first introduced in 1987, the types of studies were identified as:
• Pilot or background studies carried out in a small number of

subjects as a preliminary assessment of ADME.
• BA/BE studies.
• Pharmacokinetic studies.
• Other in vivo studies such as those using pharmacological or
clinical endpoints in humans or animals.
• In vitro studies such as dissolution and protein binding studies.
While the original focus was on in vivo studies in healthy subjects, this has
expanded to include plasma sampling in patients as part of population
pharmacokinetic studies, exposure response studies and pharmacokinetic/
pharmacodynamic studies.

There are numerous types of clinical pharmacology studies conducted

during the development of a new drug. These include both studies on
healthy subjects without the disease intended for treatment (Phase I) and
studies involving patients (Phase II and III).
Studies in healthy subjects primarily focus on safety aspects of the drug,
in establishing dose-toxicity relationships. These studies also investigate the
pharmacokinetics for the drug under development, dose proportionality,
absolute bioavailability, mass balance, effect of food, different formulations,
as well as special populations.
Studies conducted in patients primarily relate to establishing efficacy and
dose/response. In addition, optimal dosing interval, effect of severity of
disease, tolerance, and adverse reactions are determined.
One significant example from this era involved a once-a-day extended
release theophylline product which was shown to have a significant change
in bioavailability when administered with a high fat meal. This important
safety information resulted in the following precaution being added to the
product’s labeling:
Drug/Food Interactions Taking (this product) less than one hour before a high-
fat-content meal, such as 8 oz whole milk, 2 fried eggs, 2 bacon strips, 2 oz
hashed brown potatoes, and 2 slices of buttered toast (about 985 calories,
including approximately 71 g of fat) may result in a significant increase in peak
serum level and in the extent of absorption of theophylline as compared to
administration in the fasted state. In some cases (especially with doses of 900
mg or more taken less than one hour before a high-fat-content meal) serum
theophylline levels may exceed the 20mcg/mL level, above which theophylline
toxicity is more likely to occur.
A CDER Guidance [25] is available which describes current

recommendations related to food effect studies and labeling based upon
the results of such studies. Drug administration relative to meals is
sometimes of great importance. The labeling for atovaqone serves to
illustrate a situation where drug must be taken with food for optimal
efficacy:
Failure to administer (atovaquone) with meals may result in lower plasma

atovaquone concentrations and may limit response to therapy.
Era of Special Populations

With the ability to conduct pharmacokinetic studies well established,
attention advanced to additional applications. One such area was the study
of various sub-populations, including the elderly, males compared to

females and possible racial differences in pharmacokinetics. These aspects

have continued to be emphasized and currently, it is expected that all NBAs
will include analysis of data related to age, gender, and race.
CDER has used numerous methods to move forward the science of drug
regulation. This includes involvement in Workshops to discuss current drug
regulatory issues and the development of Guidances to put forward
recommendations to sponsors as to how to proceed in many areas including
clinical pharmacology studies. These Guidances include both
CDER-developed documents [26] and ICH Guidelines [27].
The importance of age-related differences in response to drugs is
discussed in a CDER Guidance [28]. A pharmacokinetic screen [29] is
recommended, consisting of obtaining blood samples from patients in Phase
II and Phase III clinical investigations. This is a means of identifying
subgroups of patients, such as the elderly, in whom the drug may have
unusual pharmacokinetic characteristics. Procedures such as the
pharmacokinetic screen have evolved into current methods of population
pharmacokinetics [30].
An example, from about 20 years ago, of a drug which proved to have
serious toxicity among some elderly patients was benoxaprofen, a non-
steroidal anti-inflammatory drug, used to treat arthritis. It was
promoted as perhaps capable of “arresting the disease process” in
rheumatoid arthritis. While it was certainly effective for labeled
indications, for certain elderly patients it was associated with fatal
cholestatic jaundice among other serious adverse reactions. If the
pharmacokinetics of benoxaprofen had been studied in the elderly, it is
possible that a dose adjustment for elderly could have been
recommended and withdrawal of benoxaprofen from the market, which
occurred in 1983, might have been avoided [31].
While for most drugs, males and females can safely receive the same dose,
for a few drugs, differences in pharmacokinetics related to gender can be
important. In 1993, the Guideline for the Study and Evaluation of Gender
Differences in the Clinical Evaluation of Drugs [32] was published. This
recommended inclusion of patients of both genders in drug development,
assessment of clinical data by gender, assessment of potential
pharmacokinetic differences between genders, and the conduct of specific
additional studies in women, when appropriate.
Patients with impaired renal or hepatic function are also important sub-
populations. Consideration of the need for dosage adjustment in
situations of renal or hepatic impairment has received considerable
attention. Guidances [33, 34] addressing these topics are available from
FDA.

Era of Drug Interactions and PK/PD Relationships

In 1991, a Workshop was held to discuss current thinking related to the
rational integration of pharmacokinetics, pharmacodynamics, and
toxicokinetics [35]. This was an important milestone along the path of
closer relationships between clinical data and pharmacokinetic data.
In CDER, a reorganization establishing the Office of Clinical
Pharmacology and Biopharmaceutics in conjunction with increased
resources related to User Fees, promoted communication among medical
reviewers and clinical pharmacology reviewers. Co-location of these
reviewers provided for increased discussions, data sharing, and
consultations.
The importance of the relationship of changes in pharmacokinetics to
drug safety and efficacy is a continuing topic of much discussion. One
related area is drug interactions, which sometimes are extremely
important.
The interaction of fluorouracil and sorivudine, which caused a
number of deaths in Japan [36] in the 1990s, served as an important
reminder of the potential consequences of drug-drug interactions.
Sorivudine was withdrawn in Japan after 15 patients who were
prescribed both sorivudine and fluorouracil died. They had developed
aplastic anemia, after taking sorivudine with fluorouracil. Knowing the
situation that had occurred in Japan, sorivudine was not approved in the
United States because of this potentially fatal drug interaction and the
fact that alternative drugs to sorivudine were available, without the
serious drug interaction potential.
Serious interactions between mibefridil and certain cholesterol lowering
“statin” drugs resulted in the removal of mibefridil from the market.
Mibefradil is a potent inhibitor of the metabolism of lovastatin and
simvastatin and if either of these drugs is taken together with mibefridil,
they can cause potentially life-threatening rhabdomyolysis related to much
higher exposure to the statin drug due to inhibited metabolism caused by
mibefridil [37].
In response to the significance of drug interactions, Guidances for the
study of potential drug interactions, both in vitro [38] and in vivo [39], are
available from FDA. Study continues on establishing in vitro/in vivo
correlations for metabolically related drug interactions, in order to increase
the predictability of in vitro drug interaction data.
An important new law went into effect in 1997. The Food and Drug
Administration Modernization Act (FDAMA) [40] contained many new
provisions including a section describing the number of required clinical
investigations needed for approval. “If the Secretary determines, based on
relevant science, that data from one adequate and well-controlled clinical

investigation and confirmatory evidence (obtained prior to or after such

investigation) are sufficient to establish effectiveness, the Secretary may
consider such data and evidence to constitute substantial evidence….” The
confirmatory evidence described can be obtained from earlier clinical trials,
pharmacokinetic data, or other appropriate scientific data. This indicates
further reliance on pharmacokinetic data in conjunction with clinical
studies in the overall development of a new drug.
Year 2000 and Onward

As we continue to move forward in the area of clinical pharmacology
aspects of drug development, we are faced with worldwide pharmaceutical
companies, an explosion of data, and increased knowledge of the
importance of optimal drug administration and the consequences of less
than optimal drug use. In this context, computer-based systems increasingly
provide an essential means of communication, as well as an effective tool for
modeling and simulation. From the internet to personal information
managers and Pocket PCs, we are nearly always close to a source of drug
information. An increasingly common utterance is that there is so much
information available but there are also increasing difficulties in sorting
through this avalanche of information to find what is useful and thereby
translating information into useful knowledge. But, there can be no
question that computer-based information will continue to expand and
progress as one of the most important means of communication and sources
of information.
Clinical trial simulation [41] has matured to a point where all available
information about a drug under development can be used efficiently to
promote more rapid drug development. The entire process of drug
development has been estimated to take up to 12 years and cost upwards of
$350 million. About one-third of this cost and half the time is spent on
clinical development. Simulation techniques can provide valuable
information related to optimal dosing schedule, expected range of response,
effects of changes in exclusion criteria on expected outcome, optimal
frequency to measure response, and the impact of compliance.
Effective labeling has become an important topic, as large amounts of
information become available for newly approved drugs. Drug interactions
studied for a new drug have implications for the other drugs involved in the
interactions and keeping labeling up to date for all drugs is a difficult task.
As difficult is the task of healthcare providers being aware of all patient
situations where dose adjustment may be appropriate, related to age,
gender, race, renal or hepatic function, or drug interactions. FDA has
proposed a new labeling format [42] in the effort to present important
dosing and other safety information more clearly and obviously.

The use of population pharmacokinetics [30] allows for the study of

differences in safety and efficacy among population subgroups. This
approach, which involves obtaining plasma samples from patients
participating in clinical studies, can permit the identification of important
factors, such as age, gender, weight, renal function, hepatic function, and
concomitant medications which can affect the safe and effective use of a
drug.
A topic of interest and considerable discussion recently is the Global
Clinical Trial. Clinical trials conducted in the United States. Europe, or
Japan often need some type of bridging study to allow the existing clinical
data to be used in the approval process in a different region of the world. A
Global Clinical Trial would include patients from the three ICH regions and
might allow the results of the trial to be directly applicable for approval in
all three regions and thereby speed worldwide drug approval.
Risk management is a frequently heard term in the current and future era
of a complex healthcare environment, with many potent new drugs being
approved, and an emerging global market. The FDAs Task Force on Risk
Management [43] has recommended that a new framework for risk
management activities is needed. The current system, which involves not
only the FDA but also pharmaceutical manufacturers, healthcare
practitioners, and patients, is more fragmented rather than part of an
integrated systems effort. One important recommendation relates to risk
confrontation, which involves community-based problem solving and
involves all stakeholders in the decision-making process. Regarding post-
marketing surveillance and risk assessment, it has been suggested that new
approaches be considered such as increasing reliance on computer-based,
perhaps global, health information databases, as well as gathering data
from identified sentinel facilities where staff are trained to recognize rapidly,
and report accurately, adverse reactions.
In conclusion, one of the most striking developments in this area over
the past 30 years has been the change from independent clinical studies
conducted in patients with the goal of determining safety and efficacy, and
independent pharmacokinetic studies conducted in healthy subjects, to the
current situation where these studies are viewed together. Over the years,
these two sources of data have become increasingly associated and utilized
together in numerous approaches to efficient drug development. By
obtaining some additional plasma samples from patients in clinical
studies, all studies in humans can be viewed as a continuum and a more
complete evaluation of a drug can be obtained. By the integration of all
available drug development data, dose can be better optimized for each
patient, thereby minimizing adverse reactions and promoting effective
treatment of diseases.

ACKNOWLEDGMENT
Dr. A.Cohen, Center for Human Drug Research, Leiden, The Netherlands
and Dr. P.Neels, Member of the Commission for Proprietary Medicinal
Products, Brussels, Belgium.
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Pharmacology during the 20th Century. J. Clin. Pharm. 2000, 40, 918–929.
2. Lathers, C.M. Lessons Learned from the Past: A Guide for the Future of Clinical
Pharmacology in the 21st Century. J. Clin. Pharm. 2000, 40, 946–966.
3. ICH Topics and Guidelines, http://www.ifpma.org/ich5.html.
4. Sitsen, J.M.A.Klinische Farmacologie: over mensen en geneesmiddelen.
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Pharmacol. 1999, 47, 11–12.
6. Sjöqvist, F. The Past, Present and Future of Clinical Pharmacology. Eur. J. Clin.
Pharmacol. 1999, 55, 553–557.
7. Bateman, N.; Maxwell, S. Career Focus. Clinical Pharmacology. BMJ 1999, 319,
S2–7219.
8. Gambardella, A.; Orsenigo, L.; Pammoli, F. Global Competitiveness in
Pharmaceuticals. A European Perspective; Report Prepared for the Directorate
General Enterprise of the European Commission, November 2000, http://
pharmacos.eudra.org.
9. European Federation for Pharmaceutical Sciences; New Safe Medicines Faster
Workshop Report, July 1, 2000, http://www.eufeps.org.
10. Lesko, L.; Rowland, M.; Peck, C.; Blaschke, T. Optimizing the Science of Drug
Development: Opportunities for Better Candidate Selection and Accelerated
Evaluation in Humans. Conference Report. European Journal of Pharmaceu-
tical Sciences 2000, 10, iv–xiv.
11. European Federation for Pharmaceutical Sciences. Newsletter, December 2002,
http://www.eufeps.org.
12. Taylor, D. Fewer New Drugs from the Pharmaceutical Industry. Editorial. BMJ
2003, 326, 408–409.
13. Rolan, P. The Contribution of Clinical Pharmacology Surrogates and Models to
Drug Development—A Critical Appraisal. Br. J. Clin. Pharmacol. 1997, 44, 219–
225.
14. Senn, S. Letters. Drug Development means Economics in the End. BMJ 2001,
322, 675.
15. McCarthy, A. Pharmacogenetics. Editorial. BMJ 2001, 322, 1007–1008.
16. Grahame-Smith, D.G. How will Knowledge of the Human Genome Affect Drug
Therapy? Br. J. Clin. Pharmacol. 1999, 47, 7–10.
17. Committee for Proprietary Medicinal Products; Note for guidance on the
investigation of drug interactions, http://www.eudra.org.

18. Jones, C.T. Call for a New Approach to the Process of Clinical Trials and Drug
Registration. BMJ 2001, 322, 920–923.
19. Soto, J. Efficiency-Based Pharmacotherapy: The New Paradigm for the 21st
Century in Medicine. Eur. J. Clin. Pharmacol. 2000, 56, 525–527.
20. Medicinal Product Agency, Sweden. About MPA http://www3.mpa.se.
21. Cameron McKenna, Andersen Consulting. Evaluation of the operation of
Community procedures for the authorization of medicinal products; Evalua-
tion carried out on behalf of the European Commission, October 2000, http://
pharmacos.eudra.org.
22. The European Agency for the Evaluation of Medicinal Products; Applications in
the Centralised Procedure 1995 to July 1999—an analysis of outcomes, March
15, 2000. The European Agency for the Evaluation of Medicinal Products, http://
www.emea.eu.int.
23. The European Agency for the Evaluation of Medicinal Products; Reform of EU
Pharmaceutical Legislation; Memo/01/267, July 18, 2001, http://
www.emea.eu.int.
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Bioavailability Section of an Application, http://www.fda.gov/cder/guidance/
old071fn.pdf.
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www.fda.gov/cder/guidance/1719dft.pdf.
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27. International Conference on Harmonization Guidelines, http://www.ifpma.org/
ich5.html.
28. FDA Guidance—Study of Drugs Likely to Be used in the Elderly, http://
www.fda.gov/cder/guidance/old040fn.pdf.
29. Sheiner, L.B.; Benet, L.Z. Premarketing Observational Studies of Population
Pharmacokinetics of New Drugs. Clin. Pharm. Ther. 1985, 38, 481–487.
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guidance/1852fnl.pdf.
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in the Clinical Evaluation of Drugs, http://www.fda.gov/cder/guidance/
old036fn.pdf.
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http://www.fda.gov/cder/guidance/1449fnl.pdf.
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Study Design, Data Analysis, and Impact on Dosing and Labeling, http://
www.fda.gov/cder/guidance/2629dft.pdf.
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in Rational Drug Development, Yacobi A. et al., Eds.; Plenum Press: New York,
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clin3.pdf.
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www.fda.gov/cder/guidance/2635fnl.pdf.
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Clinical Trials. Annu. Rev. Pharmacol. Toxicol. 2000, 40, 209–234.
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Framework; Report to the FDA Commissioner from the Task Force on Risk
Management; U.S. Department of Health and Human Services, FDA, May 1999.

3
Regulatory Bases for Clinical Pharmacology
and Biopharmaceutics Information in a New
Drug Application
Mehul Mehta and John Hunt

Within the United States, the development and marketing of products for
human use in the diagnosis, cure, mitigation, treatment, or prevention of
disease, or to affect the structure or function of the body are regulated by
legislation or law that has been enacted by the U.S. Congress. The
responsibility to interpret, promulgate and enforce congressional legislation is
given to the U.S. Food and Drug Administration (FDA) [1]. To assist in
carrying out these responsibilities, the FDA implements rules or regulations
that are published in the Federal Register (FR) then codified in the U.S. Code of
Federal Regulations (CFR). Additionally, FDA publishes guidances that are
not legally binding but are intended to provide insight and direction on how to
best satisfy legislative and regulatory requirements plus they give the most
current scientific thinking within FDA. In this chapter, key drug legislation,
relevant CFR regulations, FDA guidances and more recent International
Conference on Harmonization (ICH) guidelines that impact on, or are linked
to, or provide input as to what clinical pharmacology and biopharmaceutics
35
36 Mehta and Hunt
information should be provided in a new drug application (NDA) to support

approval of a pharmaceutical product are reviewed. The parties involved in the
ICH guidelines are regulatory authorities of Europe, Japan, and the United
States, and experts from the pharmaceutical industry in the three regions.
The reader will notice, especially during the latter part of the chapter where
individual guidances and guidelines are discussed, that there is quite a bit of
overlap between the U.S. and the ICH documents as well as within the ICH
documents. However, in the view of the authors, removing or minimizing
this overlap would be a disservice to these documents and so even at the risk
of being repetitious, regulatory basis which support clinical pharmacology
and biopharmaceutic information from all the relevant documents is
presented.
For the purpose of this chapter, clinical pharmacology is interpreted to
encompass (i) that which the body does to a drug in terms of absorption,
distribution, biotransformation and excretion (i.e., its pharmacokinetics
(PK) and exposure characteristics) and (ii) what the drug and/or its
metabolite(s) do to the body in terms of mechanism(s) of action and
resultant biochemical, physiological, and/or clinical effects or outcomes
(i.e., its pharmacodynamics (PD) or response characteristics) when
administered to healthy subjects and/or the target patient population(s) that
may include “special populations” where dose and/or dosing regimen
changes may or may not be needed. Biopharmaceutics is interpreted to
encompass the characterization of the physical and chemical properties of a
drug and/or its dosage form(s) along with determining performance
characteristics via in vitro and/or in vivo procedures or methodologies.
Often clinical pharmacology and biopharmceutics information overlap.
U.S. DRUG LEGISLATION
In the U.S., the key piece of legislation or law that sets the framework to
insure that safe and effective pharmaceutical products reach and are
maintained in the marketplace is the Federal Food, Drug and Cosmetic Act
(FDCA)1 [http://www.fda.gov/opacom/laws/fdcact/fdctoc.htm] [1]. Today’s
version of the FDCA is the culmination of numerous modifications or
amendments to the original legislation that was enacted in 1938 as the result
of deaths due to a sulfanilamide product that contained diethylene glycol or
antifreeze in the formulation. The 1938 FDCA set a requirement that safety
needed to be demonstrated for drugs and before a new drug could be
introduced into interstate commerce a new drug application (NDA) needed
to be submitted to FDA. Drug products marketed before 1938 were
however exempted from the FDCA (i.e., “grandfather drugs”).

Clinical Pharmacology and Biopharmaceutics Information 37
Historical and more current amendments to the FDCA include the

Durham-Humphrey Amendment of 1951, the Kefauver-Harris
Amendments of 1962, the Drug Listing Act of 1972, the National
Environmental Policy Act of 1974, Medical Device Amendments of 1976,
the Orphan Drug Act of 1983, the Drug Price Competition, and Patent Term
Restoration Act of 1984 (i.e., Waxman-Hatch Amendments), the Drug
Exports Amendments Act of 1986, the Prescription Drug Marketing Act of
1988, the Safe Medical Devices Act of 1990, the Prescription Drug User Fee
Act (PDUFA) of 1992, the FDA Modernization Act (FDAMA) of 1997 and
the Best Pharmaceuticals Act for Children of 2002. Of the nine chapters in
the present FDCA, the key chapters and sections related to drugs include
and address the following.
Chapter II of FDCA—Definitions (Section 201)

In this section, definitions for key terms like drug, interstate commerce,
labeling, etc. are given.
Chapter III of FDCA—Prohibited Acts and Penalties

(Sections 301–310)
Identified in these sections are different actions or scenarios that are
prohibited for drug products intended for interstate commerce (e.g.,
introduction of adulterated or misbranded products, etc.). Also identified
are the legal consequences that can occur, which include criminal charges,
monetary penalties and/or seizures if one is involved in actions or scenarios
that are defined as prohibited.
Chapter V of FDCA—Drugs and Devices (Sections 501–563)

Sections 501 and 502—Adulterated and Misbranded Drugs
Within Chapter V, Section 501 addresses when a drug shall be deemed
adulterated. It raises the fact that regulations can be promulgated to
prescribe appropriate tests or methods of assay for the determination of
strength, quality, or purity of drugs if such tests or methods are not set forth
in an official compendium (i.e., the “United States Pharmacopoeia and the
Homoeopathic Pharmacopoeia of the Unites States”). Section 502 addresses
when a drug shall be deemed misbranded.
Section 505—New Drugs

Of the different chapters and sections covered in the FDCA, it is Section 505
of Chapter V for New Drugs which sets the overall foundation or basis for

38 Mehta and Hunt
having pharmaceutical manufacturers or sponsors submit information to

FDA before a product is allowed to market. Section 505 establishes that
before the introduction of any new product into interstate commerce, an
application needs to be filed with FDA for approval. Under Sections
505(b)(1), 505(b)(2), and 505(j), three types of drug applications are
described. It is noted that Sections 505(b)(2) and 505(j) are the result of the
Drug Price Competition and Patent Term Restoration Act of 1984.
Together, these two sections replaced FDA’s paper NDA policy that
permitted an applicant to rely on studies published in the scientific literature
to demonstrate safety and effectiveness of duplicates of certain post-19622
innovator or pioneer drug products.
For an NDA that is covered under 505(b)(1), the application contains
full reports of clinical investigations of safety and effectiveness that are
conducted by or for the applicant. For an NDA covered under 505(b)(2),
one or more of the safety and effectiveness investigations used to support
the application’s approval are not conducted by or for the applicant and
the applicant has not obtained a right of reference or use from the person
by or for whom the investigations are conducted. Section 505(b)(2) allows
for the approval of products other than generic products (see below) and it
permits the use of literature or an Agency finding of safety and/or
effectiveness of a FDA-approved drug to support the approval of a
product.
In addition to safety and efficacy information. Section 505 also indicates
that 505(b)(1) and (2) applications need to provide(i) a list of the articles
used as components for the drug, (ii) a statement of the composition of the
drug, (iii) a description of the methods used in, and the facilities and controls
used for the manufacture, processing, and packing of the drug, (iv) samples
of the drug and the articles used as components if requested, and (v) samples
of the proposed labeling.
The third type of application is a 505(j) application that is also known
as an abbreviated new drug application (ANDA). The 505(j) application is
for duplicates of already approved products, or generic products, and
although it is beyond the scope of this chapter, it is noted that such an
application is to contain, among other things, information to show that
the product for approval is the same in active ingredient, dosage form,
strength, route of administration, labeling and performance characteristics
(i.e., is bioequivalent) as that of a previously approved product (i.e., the
reference listed drug or RLD), that is, unless a suitability petition is filed and
accepted, for example, for a different active ingredient in a combination
drug product, or a different dosage form, strength or route of administration
than the RLD.
If a generic product is found to be bioequivalent to the RLD and it is
approved, it will then be included in the FDA reference text entitled,

Approved Drug Products with Therapeutic Equivalence Evaluations which

is often referred to as the “Orange Book”3 [http://www.fda.gov/cder/orange/
default.htm] [2]. In this book, a generic product that is bioequivalent to the
RLD will be assigned a code of “A” which means that it can be substituted
for the RLD product or any other generic product that is approved and
coded A.
Via Section 505(i), the bases for dealing with new pharmaceuticals that
are under investigation or development prior to filing an NDA are addressed
(i.e., investigational new drug (IND) applications). This section indicates
that regulations should be promulgated to address the investigational
situation for new drugs. It further indicates that a clinical investigation for a
new drug may begin 30 days after the applicant has submitted information
about the drug and the intended clinical investigation. The information to
be provided should include a description of the design of the clinical
investigation plus information to allow an assessment of safety that is to
include “adequate information on the chemistry and manufacturing of the
drug, controls available for the drug and primary data tabulations from
animal studies or human studies.” A clinical investigation may be prevented
from being initiated during the 30-day window of time (i.e., a “clinical
hold”) if insufficient information is provided to allow for assessment of
safety considerations, or there are real safety concerns based on the
information that is provided. Following the initial IND clinical
investigation, the FDA allows subsequent IND clinical investigations to not
be restricted to the 30-day requirement before a study can be started.
However, a clinical hold can be imposed on any IND investigation before it
is started or after it is initiated if there are justified safety concerns.
Section 505A—Pediatric Studies of Drugs

As a result of the FDA Modernization Act (1997) [http://www.fda.gov/cder/
fdama], the FDCA was amended to address pediatric drug studies among
other things. If it was determined (i) for 505(b)(l) applications before a new
drug’s approval (i.e., before 2002), or (ii) for an already approved drug that
is identified on a list prepared by FDA, that information related to the use of
the drug in the pediatric population may provide health benefits to this
population, a written request could be sent to the drug manufacturer or
sponsor to conduct a pediatric study(s). Pediatric studies may only need to
include “pharmacokinetic studies,” if appropriate, as compared to the more
classical clinical safety and efficacy studies. This assumes that (i) the disease
being treated or diagnosed is similar in nature between adult and pediatric
patients, (ii) there would be a similar safety profile between adult and
pediatric patients, and (iii) there are similar PK (and PD relationships if
known) between the two populations. If a study(s) is carried out as

40 Mehta and Hunt
requested and specified by FDA, the applicant could obtain six months of
additional marketing exclusivity for an NDA. After January 1, 2002 all
newly submitted NDAs must include pediatric information if appropriate.
However, the 2002 Best Pharmaceuticals Act for Children extended the time
to allow drug sponsors to apply for six months marketing exclusivity until
October 2007 for both new NDAs or drugs on FDAs list for which pediatric
information would be important to obtain.
Section 506—Fast Track Products

To facilitate the development and to expedite the review of a drug product
for the treatment of a serious or life-threatening condition where the
product demonstrates the potential to address unmet medical needs for the
condition, Section 506 addresses this situation. The fast track approval of
such a product can be based on the determination that the product has an
effect on a clinical endpoint or on a surrogate endpoint that is reasonably
likely to predict clinical benefit. However, the approval of a fast track
product may be subject to a requirement that the sponsor conduct
appropriate postapproval studies to validate the surrogate endpoint or
otherwise confirm the effect on the clinical endpoint within a specified
time.
Section 506A—Manufacturing Changes

For manufacturing changes, they are addressed in Section 506A. This
section discusses “major” and other manufacturing changes in a general
sense and touches upon when a supplemental application to an NDA is
needed to support a change. A manufacturing change is considered a major
change if it is determined to have substantial potential to adversely affect the
identity, strength, quality, purity, or potency of the drug as they may relate
to the safety or effectiveness of the drug. Related criteria include (i) a
qualitative or quantitative formulation change for the involved drug or a
change in specifications in the approved application, (ii) the determination
by regulation or guidance that completion of an appropriate clinical study
demonstrating equivalence of the drug to the drug as manufactured without
the change is required, or (iii) a change determined by regulation or
guidance to have a substantial potential to adversely affect the safety or
effectiveness of the drug.
Sections 525 to 528—Drugs for Rare Diseases or Conditions
These sections are the result of the Orphan Drug Act of 1983. The
Pharmaceuticals that are covered are for diseases or conditions that are rare
in the United States. A “rare disease or condition” is defined as any disease

or condition that (i) affects less than 200,000 persons in the U.S. or (ii)
affects more than 200,000 persons in the U.S. for which there is no
reasonable expectation that the cost of developing and making the drug
available will be recovered from U.S. sales. This section further explains that
a manufacturer or sponsor needs to request that a drug be designated for a
rare disease or condition before the submission of an application under
Section 505(b).
For a drug that is given orphan drug status, the expectations are that
similar clinical pharmacology and biopharmaceutics information would be
provided in an NDA as that for a drug that is not given the orphan drug
status.
Chapter VII of FDCA—Fees Relating to Drugs

(Sections 735–736)
This chapter and its sections are the result of the Prescription Drug User Fee
Act of 1992. Under this part of the FDCA, fees are authorized and specified
as to what is to be charged to a drug manufacturer or sponsor who submits
a human drug application via 505(b)(1) or 505(b)(2), or as a supplement to
such an approved application. The fees are to cover the expenses that are
incurred for the review of an application. As a result of a reauthorization in
1997, fees are now not to extend past October 1, 2002 unless there is
another reauthorization.
CFR REGULATIONS
As has been previously covered, FDA is given the responsibility to interpret,
promulgate and enforce U.S. drug legislation, or more specifically the
FDCA. The FDCA, although being quite specific in some sections as to what
the intent and expectations are, other sections allow for further clarification
or interpretation of the intent, expectations and/or what is needed or
required to comply with and enforce the law. As previously noted, to assist
in carrying out its responsibilities related to the FDCA, FDA will publish
notices, proposed rules, and regulations plus finalized rules and regulations
in the FR [3] followed by codification of finalized rules and regulations in
the CFR4 [4]. For the purpose of this chapter, only highlights from parts
300.50, 312, 314, and 320 of Chapter I (Food and Drug Administration,
Department of Health and Human Services) of Title 21 (Food and Drugs) of
the CFR will be covered.
For the different CFR parts, when taking into account this chapter’s
objective of addressing the regulatory bases for needing clinical
pharmacology and biopharmaceutics information in a NDA, they will be
covered in a sequence and cross referenced as appropriate to allow for a

42 Mehta and Hunt
more interrelated perspective as needed. For complete content of the

discussed parts, readers are referred to the CFR.
21 CFR 320—Bioavailability and Bioequivalence

Requirements
Historically, part 320 that addresses bioavailability (BA) and bioequivalence
(BE) requirements was the outcome of a 1974 report that was prepared by
the Drug Bioequivalence Study Panel that was convened under the U.S.
Congress Office of Technology Assessment [5]. The charge to the panel was
to “examine the relationships between chemical and therapeutic
equivalence of drug products and to assess the capability of current
technology—short of therapeutic trials in man—to determine whether drug
products with the same physical and chemical composition produce
comparable therapeutic effects.” In the report one conclusion was that the
standards and regulatory practices at the time did not insure bioequivalence
for drug products. The report went on to make recommendations as to what
could be done. As a result, in 1977 FDA finalized its Bioavailability and
Bioequivalence Requirements via the FR which were subsequently codified
in the CFR.
Although the impetus for the BA and BE requirements was for assuring
therapeutic equivalence among duplicate or generic products, the
requirements were also crafted to establish information needs to support the
approval of NDAs for new molecular entities (NMEs) or new chemical
entities (NCEs), as well as for defined changes for already approved NDA
products. The inclusion of requirements for NDAs was to (i) foster better
product quality, (ii) define or characterize what happens to a drug and its
dosage form(s) when administered, (iii) provide information to help
understand or interpret clinical safety and efficacy findings as appropriate,
and (iv) provide useful information via the product’s labeling or package
insert for healthcare professionals.
Under Section 320.1, definitions are provided. The term bioavailability is
defined as the rate and extent to which the active ingredient or active moiety
is absorbed from a drug product and becomes available at the site of action.
It further states that for drug products that are not intended to be absorbed
into the bloodstream, bioavailability may be assessed by measurements
intended to reflect that rate and extent to which the active ingredient or
active moiety becomes available at the site of action. Other terms that are
defined include bioequivalence, drug product, pharmaceutical equivalents,
and pharmaceutical alternatives (see Glossary).
For part 320, key sections and subsections include the following, for
which some are expanded upon as needed.

• 320.21 Requirements for submission of in vivo bioavailability

and bioequivalence data.
Under this section, as related to NDAs, it indicates that “Any
person submitting a full new application to the FDA shall include
in the application either:
1. Evidence demonstrating the in vivo bioavailability of the drug
product that is the subject of the application; or
2. Information to permit FDA to waive the submission of
evidence demonstrating in vivo bioavailability.”
This section goes on to indicate that any person submitting a
supplemental application to FDA shall include in the
supplemental application evidence demonstrating the in vivo
bioavailability of the product or information to permit FDA to
waive the submission of evidence demonstrating in vivo
bioavailability for changes that include:
1. A change in the manufacturing process, including a change in
product formulation or dosage strength, beyond the
variations provided for in the approved application.
2. A change in the labeling to provide for a new indication for
use of the drug product, if clinical studies are required to
support the new indication for use.
3. A change in the labeling to provide for a new dosage regimen
or for an additional dosage regimen for a special patient
population, e.g., infants, if clinical studies are required to
support the new or additional dosage regimen.
• 320.22 Criteria for waiver of evidence of in vivo bioavailability
or bioequivalence.
• 320.23 Basis for demonstrating in vivo bioavailability or
bioequivalence.
• 320.24 Types of evidence to establish bioavailability or
bioequivalence.
This section covers the different types of in vivo and in vitro
methods that can be used to determine bioavailability and
bioequivalence. They are ranked in descending order of
accuracy, sensitivity and reproducibility as stated or summarized
as follows:
1. i. An in vivo test in humans in which the concentration of

the active ingredient or active moiety, and, when
appropriate, its active metabolite(s), in whole blood,

44 Mehta and Hunt
plasma, serum, or other appropriate biological fluid is

measured as a function of time,
ii. An in vitro test that has been correlated with and is
predictive of human bioavailability data; or
iii. An in vivo test in animals that has been correlated with
and is predictive of human bioavailability data.
2. An in vivo test in humans in which the urinary excretion of the

active moiety, and, when appropriate, its active metabolite(s),
are measured as a function of time.
3. An in vivo test in humans in which an appropriate acute
pharmacological effect of the active moiety, and, when
appropriate, its active metabolite(s), are measured as a
function of time if such effect can be measured with sufficient
accuracy, sensitivity, and reproducibility.
4. Well-controlled clinical trials in humans that establish the
safety and effectiveness of the drug product, for purposes of
establishing bioavailability, or appropriately designed
comparative clinical trials, for purposes of establishing
bioequivalence.
5. A currently available in vitro test acceptable to FDA (usually a
dissolution rate test) that ensures human in vivo bioavailability.
6. Any other approach deemed adequate by FDA to establish
bioavailability and bioequi valence.
• 320.25 Guidelines for the conduct of an in vivo bioavailability
study.
Subheadings for the subsections under this section include:
a. Guiding principles.
b. Basic design.
c. Comparison to a reference material.
d. Previously unmarketed active drug ingredients or therapeutic
moieties.
e. New formulations of active drug ingredients or therapeutic
moieties approved for marketing.
f. Controlled release formulations.
g. Combination drug products.
h. Use of a placebo as the reference material.
i. Standards for test drug product and reference material.
Related to subsection (d) that addresses previously unmarketed
active drug ingredients or therapeutic moieties, it states that the

purpose of an in vivo bioavailability study is to determine the

bioavailability of the formulation proposed for marketing as
well as to determine essential pharmacokinetic characteristics of
the active drug ingredient or therapeutic moiety such as rate of
absorption, extent of absorption, half-life, excretion,
metabolism, and dose proportionality. It further indicates that
such characterization is a necessary part of the investigation of
the drug to support drug labeling.
Under the umbrella to support drug labeling as outlined in this
subsection, and with the experience that has been obtained over
time since implementation of the BA and BE Requirements,
along with advances in technology, updated and added
information needs, in the realm of clinical pharmacology and
biopharmaceutics (as defined above and under the purview of 21
CFR 320), are being asked to be addressed by sponsors in their
drug development programs for new products. As will be
covered in the section that discusses FDA guidances, FDA
provides more current thinking on such information needs as
related to the different aspects of clinical pharmacology and
biopharmaceutics. (Note: Likewise in ICH guidelines, they too
present and expand upon information needs in the areas of
clinical pharmacology and biopharmaceutics for drug product
registration, most of which is consistent with FDA guidances.)
• 320.26 Guidelines on the design of a single-dose in vivo
bioavailability study.
• 320.27 Guidelines on the design of a multiple-dose in vivo
bioavailability study.
21 CFR 300.50—Combination Drugs

Under this CFR part it addresses fixed-combination prescription drugs for
humans. It states that “Two or more drugs may be combined in a single
dosage form when each component makes a contribution to the claimed
effects and the dosage of each component (amount, frequency, duration) is
such that the combination is safe and effective for a significant patient
population requiring such concurrent therapy as defined in the labeling for
the drug.” It further explains that special cases of this general rule are where
a component is added (i) to enhance the safety or effectiveness of the
principal active component and (ii) to minimize the potential for abuse of
the principal active ingredient.

46 Mehta and Hunt
Related to 21 CFR 300.50 from a clinical pharmacology and

biopharmaceutics perspective, specifically for the scenario where the new
combination product is to be administered as an alternative to giving two or
more currently marketed, single ingredient products, one is referred to 21
CFR 320.25 (g) as identified above. Here it indicates that an in vivo
bioavailability study is needed to determine if the rate and extent of
absorption of each active drug ingredient or therapeutic moiety of the
combination product is equivalent to the rate and extent of absorption of
each active drug ingredient or therapeutic moiety administered concurrently
in separate single-ingredient preparations. Information to address drug-
drug interaction implications for the two or more drugs in a combination
product is also usually needed.
21 CFR 312—Investigational New Drug Application
Within 21 CFR 312, some of what is presented is addressed in Section 505(i)

of Chapter V of the FDCA as covered above. However, within 21 CFR 312
expanded and more detailed information related to INDs is given (e.g.,
information related to IND content and format, type of IND amendments
and reports, administrative related actions, responsibilities of sponsors and
investigators, etc.).
Of note, under Section 312.21, it indicates that the clinical investigation
of a previously untested drug is generally divided into three phases (Phases
1, 2, and 3). In general the phases are carried out sequentially but they may
overlap.
Phase 1 is where the initial introduction of an investigational new drug
into humans occurs. The studies in Phase 1 are designed to determine the
metabolism and pharmacologic actions of the drug, side effects associated
with increasing doses and, if possible, obtain early evidence of effectiveness.
Ideally, sufficient information about the drug’s pharmacokinetics and
systemic exposure plus pharmacological effects or pharmacodynamics
should be obtained to permit the design of well-controlled, scientifically
sound Phase 2 studies. The number of subjects or patients used in Phase 1
studies can vary with the drug but is usually in the range of 20–80.
Phase 2 is where well-controlled clinical studies are conducted to evaluate
the effectiveness of the drug for a particular indication or indications in
patients with the disease or condition under study. Also determined are the
short-term side effects or risks associated with the drug or product. The
number of patients used in Phase 2 studies is usually no more than several
hundred.
Phase 3 studies include controlled and uncontrolled trials that are
intended to gather additional information about effectiveness and safety for

evaluating the drug’s overall benefit-risk relationship and to provide

adequate information for labeling. Phase 3 studies can include from several
hundred to thousands of patients.
Ultimately when an NDA is submitted to FDA, it includes all of the
studies that have been carried out in Phases 1, 2, and 3. Human clinical
pharmacology and biopharmaceutics information is most often obtained
from studies that are conducted as Phase 1 type studies, but with the
advent of important and useful ways to analyze and model PK and PD
data, including population PK and PD statistical approaches,
information can and is being obtained in Phase 2 and 3 studies. There are
FDA and ICH guidances and guidelines summarized below, which give
insight into this.
Lastly, in Section 312.85 there is discussion on Phase 4 studies. At the time
FDA is considering giving an NDA approval it may, with concurrence from
the NDA sponsor, request that an additional postmarketing study or studies
be conducted to delineate additional information about the drug’s risks,
benefits, and optimal use. Phase 4 type studies can be and are requested to
obtain additional clinical pharmacology- or biopharmaceuticsrelated
information if warranted.
21 CFR 314—Applications for FDA Approval to Market a New

Drug or an Antibiotic Drug
Like for 21 CFR 312, some of what is covered in 21 CFR 314 as related to
applications for market approval for a new drug is also covered in Sections
505(b) and (j) of Chapter V of the FDCA. However, 21 CFR 314 is much
more expansive and specific in addressing NDAs (and AND As) as to the
procedures and requirements for the submission to, and for the review by
FDA of such applications for approval. Also addressed are amendments,
supplements, and postmarketing reports to applications.
Under 314.2 it states that the purpose of 21 CFR 314 is to establish an
efficient and thorough drug review process in order to (i) facilitate the
approval of drugs shown to be safe and effective and (ii) ensure the
disapproval of drugs not shown to be safe and effective. Additionally, it
addresses the establishment of a system for FDAs surveillance of marketed
drugs. Via Section 314.50 it covers the content and format of an NDA
application that is to include summary sections and technical sections for
the areas of (i) chemistry, manufacturing, and controls, (ii) nonclinical
pharmacology and toxicology, (iii) human pharmacokinetics and
bioavailability, (iv) microbiology, and (v) clinical data along with statistical
analyses.
For clinical pharmacology and biopharmaceutics related information,
314.50(d)(3) indicates that a technical section should include human

48 Mehta and Hunt
pharmacokinetic data and human bioavailability data, or information

supporting a waiver of the submission of in vivo bioavailability data as
covered under 21 CFR 320. Further it indicates that a description of each
of the human pharmacokinetic and bioavailability studies performed by
or on behalf of the applicant should be provided along with a description
of the analytical and statistical methods used plus a statement related to
informed consent procedures used per study. Additionally, if the
application describes—in the chemistry, manufacturing, and controls
sections—specifications or analytical methods needed to assure the
bioavailability of the drug product or drug substance, or both, a
statement of the rationale for establishing the specifications or analytical
methods, including data and information supporting the rationale
should be provided. Lastly, it is indicated that there should be
summarizing discussion and analysis of the pharmacokinetics and
metabolism of the active ingredients and the bioavailability or
bioequivalence, or both, of the drug product. In addition to what is
covered in 21 CFR 314, 21 CFR 320 plus FDA guidances and ICH
guidelines should additionally be consulted to get further insight as to
what specific clinical pharmacology and biopharmaceutics information
and data should be provided in an NDA.
FDA GUIDANCES
Like the FR and CFR that are often used to better clarify or define the intent,
expectations, or what is needed or required to comply with or enforce the
FDCA, FDA, as already noted, prepares and publishes guidances that
provide further insight, direction, and the Agency’s current thinking on how
to best satisfy the FDCA and FR/CFR rules or regulations, albeit that FDA
guidances are not legally binding. FDA guidances also attempt to establish
uniformity and consistency as to what is needed in NDAs for submission.5
Key FDA guidances [http://www.fda.gov/cder/guidance] that address
different aspects of clinical pharmacology and biopharmaceutics, as
previously defined, are covered.
Please note that only the guidances that are posted as “final” on the
CDER web page are summarized below and the reader is encouraged to
look up guidances that are posted but are at the “draft” stage. Additionally,
several of these “final” guidances deal with either a particular drug product
or a specific therapeutic area and therefore are not considered in this
chapter; only the “final” guidances that cover the general, broad-based
principles which apply to majority of the drug products and therapeutic
areas are summarized below.

Clinical Pharmacology
“Format and Content of the Human Pharmacokinetics and

Bioavailability Section of an Application” Guidance (1997)
This guidance is actually a reissuance of the guideline with the same title
that was issued in 1987 and is intended to assist applicants to prepare the
Human Pharmacokinetics and Bioavailability section of an NDA. After
providing a brief overview of what types of studies are generally expected
for NDAs, the guidance provides the outline of format for this section. The
section should contain, in a tabular presentation, a summary of the
studies, data, and overall conclusions, drug formulation, analytical
methods, and a product in vitro release method (e.g., dissolution) if
appropriate. The tabular format, with columns identifying specific
variables for each of these components, is provided in the appendix.
Finally, individual study report format and other considerations are
covered. It should be noted that even though the guideline was created
almost 15 years ago, this is an excellent document and the formatting
recommendations conveyed here are followed, as a minimum, to date by
most applicants.
For last several years, there has been a lot of activity and extremely
thoughtful efforts at the ICH level and a recently issued ICH guideline called
the common technical document (CTD) provides an expanded and updated
version of this guideline. This and other relevant ICH documents are
covered later in the chapter.
“Guideline for the Study of Drugs Likely to be used in the

Elderly” (1989)
Even though written 12 years ago with the primary intent to advice sponsors
on how to undertake clinical investigation of drugs likely to be used in the
elderly, this guideline is a milestone in terms of identifying, explaining, and
recommending clinical pharmacology studies in terms of drug-drug
interactions, drug-disease interactions, special populations (elderly, renally
impaired and hepatically impaired), and pharmacodynamic studies (in the
elderly). Further, this guideline also established the concept of
“Pharmacokinetic Screen” which has subsequently matured into the science
of “Population Pharmacokinetics.” In view of the authors, this is a must-
read classical document. Not surprisingly, this is also one of the first topics
that were finalized at the ICH and in view of the authors, the E7 document,
namely “Clinical Trials in Special Populations—Geriatrics” is an excellent
update of this ’89 document. The E7 document is covered in detail later on
in the chapter.

50 Mehta and Hunt
“Drug Metabolism/Drug Interaction Studies in the Drug

Development Process: Studies In Vitro” Guidance (1998)
This guidance is directed towards a broad class of drugs, namely molecules
with a molecular weight below 10 kilo Daltons, and it provides suggestions
on current approaches to in vitro studies of metabolism and interactions of
such molecules. The guidance is intended to encourage routine, thorough
evaluation of metabolism and interactions in vitro whenever feasible and
appropriate. This guidance recognizes that the importance of such an
approach will vary depending on the drug in development and its intended
clinical use. It also recognizes that clinical observations can address some of
the same issues identified in this document as being susceptible to in vitro
study.
The guidance covers the following topics: observations and
conclusions; techniques and approaches for in vitro studies for drug
metabolism and drug-drug interactions (DDI); correlations between
studies in vitro and in vivo; timing of metabolism studies; labeling; and
related applications and considerations. This subject is discussed in detail
in Chapter 6 of this book.
“In Vivo Drug Metabolism/Drug Interaction Studies—Study

Design, Data Analysis, and Recommendations for Dosing
and Labeling” Guidance (1999)
This guidance provides recommendations to sponsors of NDAs and
biologies license applications (BLAs) for therapeutic biologies (hereafter
drugs) who intend to perform in vivo drug metabolism and metabolic
drug-drug interaction studies. The guidance reflects the Agency’s current
view that the metabolism of an investigational new drug should be defined
during drug development and that its interactions with other drugs should
be explored as part of an adequate assessment of its safety and
effectiveness. For metabolic drug-drug interactions, the approaches
considered in the guidance are offered with the understanding that
whether a particular study should be performed will vary, depending on
the drug in development and its intended clinical use. Furthermore, not
every drug-drug interaction is metabolism-based, but may arise from
changes in PK caused by absorption, tissue, and/or plasma binding,
distribution and excretion interactions. Drug interactions related to
transporters or pharmacodynamic-based drug interactions are not
covered in this guidance.
After a brief discussion on metabolism and metabolic DDIs, the guidance
covers the following topics: general strategies; design of in vivo metabolic
drug-drug interaction studies; and labeling.

“Pharmacokinetics in Patients with Impaired Renal

Function—Study Design, Data Analysis, and Impact
on Dosing and Labeling” Guidance (1998)
This guidance is intended for sponsors who, during the investigational phase
of drug development, plan to conduct studies to assess the influence of renal
impairment on the PK of an investigational drug. Topics covered in this
guidance are: deciding whether to conduct a study in patients with impaired
renal function (when studies may be important, when studies may not be
important); study design (basic “full” study design, reduced/staged study
design, population PK studies, effect of dialysis on PK, PD assessments);
data analysis (parameter estimation, modeling the relationship between renal
function and PK, development of dosing recommendations); and labeling
(clinical pharmacology, precautions/warnings, dosage and administration,
overdosage).
“Population Pharmacokinetics” Guidance (1999)

This guidance makes recommendations on the use of population PK in the
drug development process to help identify differences in drug safety and
efficacy among population subgroups. It summarizes scientific and
regulatory issues that should be addressed using population PK. The
guidance discusses when to perform a population PK study and/or analysis;
how to design and execute a population PK study; how to handle and
analyze population PK data; what model validation methods are available;
and how to provide appropriate documentation for population PK reports
intended for submission to the FDA.
“Pharmacokinetics in Patients with Impaired Hepatic

Function: Study Design, Data Analysis, and Impact on
Dosing and Labeling” Guidance (2002)
This guidance provides recommendations to sponsors planning to conduct
studies to assess the influence of hepatic impairment on the PK and, where
appropriate, PD of drugs or therapeutic biologies. This guidance addresses:
when studies are and may not be recommended; the design and conduct of
studies to characterize the effects of impaired hepatic function on the PK of
a drug; characteristics of patient populations to be studied; and analysis,
interpretation, and reporting of the results of the studies and description of
the results in labeling.

52 Mehta and Hunt
Biopharmaceutics
“Bioanalytical Method Validation” Guidance (2001)
This guidance provides assistance to sponsors of INDs, NDAs, AND As,
and supplements in developing bioanalytical method validation information
used in human clinical pharmacology, BA, and BE studies requiring PK
evaluation. This guidance also applies to bioanalytical methods used for
nonhuman pharmacology/toxicology studies and preclinical studies. For
studies related to the veterinary drug approval process, this guidance applies
only to blood and urine BA, BE, and PK studies. The information in this
guidance generally applies to bioanalytical procedures such as gas
chromatography (GC), high-pressure liquid chromatography (LC),
combined GC and LC mass spectrometric (MS) procedures such as LC-MS,
LC-MS-MS, GC-MS, and GC-MS-MS performed for the quantitative
determination of drugs and/or metabolites in biological matrices such as
blood, serum, plasma, or urine. This guidance also applies to other
bioanalytical methods, such as immunological and microbiological
procedures, and to other biological matrices, such as tissue and skin
samples. The guidance touches upon the full, partial, and cross validation
and then covers the following topics in detail: reference standard; method
development (chemical as well as microbiological and ligand-binding
assays); application of validated method to routine drug analysis; and
documentation.
“Dissolution Testing of Immediate Release Solid Oral Dosage

Forms” Guidance (1997)
This guidance is intended to provide (i) general recommendations for
dissolution testing; (ii) approaches for setting dissolution specifications
related to the biopharmaceutic characteristics of the drug substance; (iii)
statistical methods for comparing dissolution profiles; and (iv) a process to
help determine when dissolution testing is sufficient to grant a waiver for an
in vivo bioequivalence study. This document also provides recommendations
for dissolution tests to help ensure continuous drug product quality and
performance after certain postapproval manufacturing changes.
Information on dissolution methodology, apparatus, and operating
conditions for dissolution testing of IR products is provided in summary
form in Appendix A. This guidance is intended to complement the SUPAC—
IR guidance for industry (Immediate Release Solid Oral Dosage Forms:
Scaleup and Post-Approval Changes: Chemistry, manufacturing and
Controls, In Vitro Dissolution Testing, and In Vivo Bioequivalence
Documentation) with specific reference to the generation of dissolution
profiles for comparative purposes.

The topics covered in this guidance are: biopharmaceutics classification

system; setting dissolution specifications; dissolution profile comparisons;
dissolution and SUPAC-IR; and biowaivers.
“Extended Release Oral Dosage Forms: Development,

Evaluation, and Application of in vitro/in vivo Correlations”
Guidance (1997)
This guidance provides recommendations to pharmaceutical sponsors who
intend to develop documentation in support of an in vitro/in vivo
correlation (IVIVC) for an oral extended release (ER) drug product for
submission in an NDA or ANDA. The guidance presents a comprehensive
perspective on (i) methods of developing an IVIVC and evaluating its
predictability; (ii) using an IVIVC to set dissolution specifications; and (iii)
applying an IVIVC as a surrogate for in vivo bioequivalence when it is
necessary to document bioequivalence during the initial approval process or
because of certain pre or postapproval changes (e.g., formulation,
equipment, process, and manufacturing site changes).
The topics covered in this guidance are: categories of in vitro/in vivo
correlations; general considerations; development and evaluation of a level
A in vitro/in vivo correlation; development and evaluation of a level C
correlation; and applications of an IVIVC.
“Waiver of In Vivo Bioavailability and Bioequivalence Studies for

Immediate-Release Solid Oral Dosage Forms Based on a
Biopharmaceutics Classification System” Guidance (2000)
This guidance provides recommendations for sponsors of INDs, NDAs,
ANDAs, and supplements to these applications who wish to request a
waiver of in vivo BA and/or BE studies for IR solid oral dosage forms. These
waivers are intended to apply to (i) subsequent in vivo BA or BE studies of
immediate-release (IR) formulations after the initial establishment of in vivo
BA during the IND phase and (ii) in vivo BE studies of IR oral dosage forms
in ANDAs. In addition to the regulations at 21 CFR 320 that address
biowaivers, this guidance explains when biowaivers can be requested for IR
solid oral dosage forms based on an approach termed the Biopharmaceutics
Classification System (BCS).
The topics covered in this guidance are: the biopharmaceutics classification
system; methodology for classifying a drug substance and for determining
the dissolution characteristics of a drug product; additional considerations
for requesting a biowaiver; regulatory applications of the BCS; and data to
support a request for biowaivers.

54 Mehta and Hunt
“Statistical Approaches to Establishing Bioequivalence”

Guidance (2001)
This guidance provides recommendations to sponsors and applicants who
intend, either before or after approval, to use equivalence criteria in
analyzing in vivo or in vitro BE studies for INDs, NDAs, ANDAs, and
supplements to these applications. This guidance discusses three approaches
for BE comparisons: average, population, and individual. The guidance
focuses on how to use each approach once a specific approach has been
chosen. This guidance replaces a prior FDA guidance entitled Statistical
Procedures for Bioequivalence Studies Using a Standard Two-Treatment
Crossover Design, which was issued in July 1992.
The topics covered in this guidance are: statistical model; statistical
approaches for bioequivalence; study design; statistical analysis; and
miscellaneous issues.
“Bioavailability and Bioequivalence Studies for Orally Administered

Drug Products—General Considerations” Guidance (2000)
This guidance is intended to provide recommendations to sponsors or
applicants planning to include BA and BE information for orally
administered drug products in the INDs, NDAs, ANDAs, and their
supplements. This guidance addresses how to meet the BA and BE
requirements set forth in 21 CFR 320 as they apply to dosage forms
intended for oral administration. These include tablets, capsules, solutions,
suspensions, conventional/immediate release, and modified (extended/
delayed) release drug products. The guidance is also generally applicable to
nonorally administered drug products where reliance on systemic exposure
measures is suitable to document BA and BE (e.g., transdermal delivery
systems and certain rectal and nasal drug products).
This guidance starts with the definitions and a detailed discussion of the
terms BA and BE which is then followed by a discussion on the following
topics: methods to document BA and BE; comparison of BA measures in BE
studies; documentation of BA and BE; and special topics namely food-effect
studies, moieties to be measured, long half-life drugs, first point Cmax,
orally administered drugs intended for local action and narrow therapeutic
range drugs.
This guidance is designed to reduce the need for FDA drug-specific BA/BE
guidances. As a result, this guidance replaces a number of previously issued
FDA drug-specific guidances which are listed in the Appendix 1 of this
guidance.
A concluding remark on the U.S. regulations and guidances is that there
are a few pertinent guidances which are at the draft stage that are not

covered in this chapter and the reader is strongly encouraged to get familiar
with them and follow their progress till issuance of the final version.
Probably the most critical ones are the “Exposure-Response” and the
“Food-Effect” guidances.
ICH GUIDELINES
With the globalization of the pharmaceutical industry, efforts have been

underway since 1990 to standardize drug applications in terms of content
and format such that an application can be registered in different countries
without being subjected to different registration requirements among
countries. Via efforts that include the participation of the European Union,
Japan, and the United States, ICH guidelines have been prepared or are in
the process of being finalized on the topics of Quality (the Q series of
guidelines), Safety (the S series of guidelines), Efficacy (the E series of
guidelines), and Multidisciplinary (the M series of guidelines). Care has been
taken while reaching consensus with the other world bodies that the
information that is needed is based on U.S. laws and CFR regulations plus
similar considerations for the other world regulatory agencies. Relevant
ICH guidelines [http://www.ifpma.org/ichl] as related to this chapter which
are either completed or at advanced stages of completion (step 4) are
covered.6
The order of presentation of these guidelines is based on their completion
dates (earliest to latest) and not the sequence number given by the ICH (e.g.,
E3 followed by E4, etc.). The reason is that it appears that clinical
pharmacology and biopharmaceutic concepts, and related recommendations,
got introduced in the earliest guidelines in a broad and diffused sense and
they subsequently got elaborated upon and covered in more detail in later
guidelines.
E7: “Studies in Support of Special Populations: Geriatrics”

Guideline (1993)
As stated earlier, it appears that this guideline is modeled after an updated
version of, the U.S. “elderly” guidance of 1989. It covers PK studies (formal
or a PK screen) in the elderly as well as renally or hepatically impaired
patients, PD/Dose-response studies and drug-drug interaction studies as
follows.
Pharmacokinetic Studies
The guideline states that most of the recognized important differences
between younger and older patients have been pharmacokinetic differences,

56 Mehta and Hunt
often related to impairment of excretory (renal or hepatic) function or to

drug-drug interactions. It is important to determine whether or not the
pharmacokinetic behavior of the drug in elderly subjects or patients is
different from that in younger adults and to characterize the effects of
influences, such as abnormal renal or hepatic function, that are more
common in the elderly even though they can occur in any age group.
Information regarding age-related differences in the pharmacokinetics of
the drug can come, at the sponsor’s option, either from a Pharmacokinetic
Screen or from formal pharmacokinetic studies, in the elderly and in
patients with excretory functional impairment.
The guideline recognizes that for certain drugs and applications (e.g.,
some topically applied agents, some proteins) technical limitations such as
low systemic drug levels may preclude or limit exploration of age-related
pharmacokinetic differences.
Pharmacokinetics in Renally or Hepatically Impaired Patients

As stated in the guideline, renal impairment is an aging-associated finding
that can also occur in younger patients. Therefore, it is a general principle
that drugs excreted (parent drug or active metabolites) significantly through
renal mechanisms should be studied to define the effects of altered renal
function on their pharmacokinetics. Such information is needed for drugs
that are the subject of this guideline but it can be obtained in younger
subjects with renal impairment.
Similarly, drugs subject to significant hepatic metabolism and/or
excretion, or that have active metabolites, may pose special problems in the
elderly. Pharmacokinetic studies should be carried out in hepatically
impaired young or elderly patient volunteers.
If a Pharmacokinetic Screen approach is chosen by the sponsor, and if
patients with documented renal impairment or hepatic impairment
(depending on the drug’s elimination pattern) are included and the results
indicate no medically important pharmacokinetic difference, that
information may be sufficient to meet this geriatric guideline’s purpose.
Pharmacodynamic/Dose Response Studies

The guideline states that the number of age-related pharmacodynamic
differences (i.e., increased or decreased therapeutic response, or side effects,
at a given plasma concentration of drug) discovered to date is too small to
necessitate dose response or other pharmacodynamic studies in geriatric
patients as a routine requirement. Separate studies are, however, recommended
in the following situations:

• Sedative/hypnotic agents and other psychoactive drugs or drugs

with important CNS effects, such as sedating antihistamines.
• Where subgroup comparisons (geriatric versus younger) in the
Phase 2/3 clinical trials database indicate potentially medically
significant age-associated differences in the drug’s effectiveness
or adverse reaction profile, not explainable by PK differences.
Drug-Drug Interaction Studies
As per the guideline, such interactions are of particular importance to
geriatric patients, who are more likely to be using concomitant medications
than younger patients, but of course are not limited to this age group.
Therefore it is a general principle, not specific to these guidelines, that in
cases where the therapeutic range (i.e., a range of toxic to therapeutic doses)
of the drug or likely concomitant drugs is narrow, and the likelihood of the
concomitant therapy is great, that specific drug-drug interaction studies be
considered. The studies needed must be determined case-by-case, but the
following are ordinarily recommended:
• Digoxin and oral anticoagulant interaction studies, because so

many drugs alter serum concentrations of these drugs, they are
widely prescribed in the elderly, and they have narrow
therapeutic ranges.
• For drugs that undergo extensive hepatic metabolism,
determination of the effects of hepatic-enzyme inducers (e.g.,
phenobarbital) and inhibitors (e.g., cimetidine).
• For drugs metabolized by cytochrome P-450 enzymes, it is
critical to examine the effects of known inhibitors, such as
quinidine (for cytochrome P-450 2D6) or ketoconazole and
macrolide antibiotics (for drugs metabolized by cytochrome P-
450 3A4). There is a rapidly growing list of drugs that can
interfere with other drugs via metabolism, and sponsors should
remain aware of it.
• Interaction studies with other drugs that are likely to be used
with the test drug (unless important interactions have been ruled
out by a Pharmacokinetic Screen).
E4: “Dose-Response Information to Support Drug

Registration” Guideline (Step 4; 1994)
This guideline covers the following topics: (i) introduction (purpose of
doseresponse information, use of dose-response information in choosing
doses, use of concentration-response data, problems with titration designs,
interaction between dose-response and time), (ii) obtaining dose-response

58 Mehta and Hunt
information (dose-response assessment should be an integral part of drug

development, studies in life-threatening diseases, regulatory considerations
when dose-response data are imperfect, examining the entire database for
dose-response information), (iii) study designs for assessing dose-response
(general, specific trial designs), and (iv) guidance and advice.
The reader is strongly encouraged to read this guideline since it lays out
the fundamental value and benefit of the exposure (i.e., dose and/or
concentration)—response information in drug development and evaluation,
and recognizes past inadequacies as well as practical limitations in
generation of this information base. As per the guideline, where a drug can
be safely and effectively given only with blood concentration monitoring,
the value of concentration-response information is obvious. In other cases,
an established concentration-response relationship is often not needed, but
may be useful for ascertaining the magnitude of the clinical consequences of
(i) pharmacokinetic differences, such as those due to drug-disease (e.g., renal
failure) or drug-drug interactions, or (ii) for assessing the effects of the
altered pharmacokinetics of new dosage forms (e.g., controlled release
formulation) or new dosage regimens without need for additional clinical
data, where such assessment is permitted by regional regulations.
Prospective randomized concentration-response studies are critical to
defining concentration monitoring therapeutic “windows” but are also
useful when pharmacokinetic variability among patients is great; in this
case, a concentration-response relationship may in principle be discerned in
a prospective study with a smaller number of subjects than could be the dose
response relationship in a standard dose-response study. Note that
collection of concentration-response information does not imply that
therapeutic blood level monitoring will be needed to administer the drug
properly. Concentration-response relationships can be translated into
doseresponse information. Alternatively, if the relationships between
concentration and observed effects (e.g., an undesirable or desirable
pharmacologic effect) are defined, patient response can be titrated without
the need for further blood level monitoring. Concentration-response
information can also allow selection of doses (based on the range of
concentrations they will achieve) most likely to lead to a satisfactory
response.
E3: “Structure and Content of Clinical Study Reports”

Guideline (Step 4; 1995)
The relevant portions of this guideline from a clinical pharmacology
perspective are the sections which cover the “drug concentration
measurements,” “drug dose, drug concentration, and relationships to
response,” and “drug-drug and drug-disease interactions” topics.

Further discussion of this guideline is not undertaken in this chapter since

these topics are also covered in other guidelines, particularly the M4
guideline discussed later in this chapter.
E8: “General Considerations for Clinical Trials” Guideline

(1997)
This guideline goes over general principles of clinical trials in terms of
protection of subjects and scientific approach in design and analysis, as well
as development methodology in terms of considerations for the
development plan and considerations for individual clinical trials.
A very informative section in this guideline is Table 1 that provides an
approach to classifying clinical studies according to objectives. The table
breaks down the types of studies into four categories, namely Human
Pharmacology, Therapeutic Exploratory, Therapeutic Confirmatory, and
Therapeutic Use and lists the objectives of such studies along with examples.
The first two categories of studies identify clinical pharmacology studies.
The Human Pharmacology category comprises studies that assess tolerance,
define/describe PK and PD, explore drug metabolism and drug interactions,
and enzyme activity. Examples of such studies are dose-tolerance studies,
single and multiple dose PK and/or PD studies, and drug interaction studies.
Similarly, the Therapeutic Exploratory category consists of studies that
explore use for the targeted indication, estimate dosage for subsequent
studies, provide basis for confirmatory study design, endpoints, and
methodologies. Examples of such studies are the earliest trials of relatively
short duration in well-defined narrow patient populations, using surrogate
or pharmacological endpoints of clinical measures, and dose-response
exploration studies.
Additional sections outlining clinical pharmacology and biopharmaceutic
considerations are:
• Quality of investigational medicinal products

• Phase I (Most typical kind of study: human pharmacology)
• Estimation of initial safety and tolerability
• Pharmacokinetics
• Assessment of pharmacodynamics
• Early measurement of drug activity
• Special considerations
• Studies of drug metabolites
• Drug-drug interactions
• Special populations
• Investigations in nursing women

60 Mehta and Hunt
E5: “Ethnic Factors in the Acceptability of Foreign Clinical

Data” Guideline (Step 4; 1998)
This guideline is based on the premise that it is not necessary to repeat an
entire clinical drug development program in a new region, and it is intended
to recommend strategies for accepting foreign clinical data as full or partial
support for approval of an application in a new region. It is a strong
endorsement of the utility of clinical pharmacology information. A couple
of key concepts—bridging study and compounds sensitive to ethnic
factors—in this guideline are based on, or utilize, clinical pharmacology
information. Additionally, it also provides a definition of a PK study, a PD
study, and Population PK Methods as well as providing a good discussion of
PK, PD, and dose-response considerations.
Bridging Study
A bridging study is defined as a supplemental study performed in the new
region to provide pharmacodynamic or clinical data on efficacy, safety,
dosage, and dose regimen in the new region that will allow extrapolation of
the foreign clinical data to the new region. Such studies could include
additional pharmacokinetic information.
Compounds Sensitive to Ethnic Factors
A compound who’s pharmacokinetic, pharmacodynamic, or other
characteristics suggest the potential for clinically significant impact by
intrinsic and/or extrinsic ethnic factors [covered further in the M4 guideline]
on safety, efficacy, or dose response.
Pharmacokinetic Study
A study of how a medicine is handled by the body, usually involving
measurement of blood concentrations of drug and its metabolite(s)
(sometimes concentrations in urine or tissues) as a function of time.
Pharmacokinetic studies are used to characterize absorption, distribution,
metabolism, and excretion of a drug, either in blood or in other pertinent
locations. When combined with pharmacodynamic measures (a PK/PD
study) it can characterize the relation of blood concentrations to the extent
and timing of pharmacodynamic effects.
Pharmacodynamic Study
A study of a pharmacological or clinical effect of the medicine in individuals
to describe the relation of the effect to dose or drug concentration. A
pharmacodynamic effect can be a potentially adverse effect (anticholinergic
effect with a tricyclic), a measure of activity thought related to clinical

benefit (various measures of beta-blockade, effect on ECG intervals,

inhibition of ACE or angiotensin I or II response), a short-term desired
effect, often a surrogate endpoint (blood pressure, cholesterol), or the
ultimate intended clinical benefit (effects on pain, depression, sudden
death).
Population Pharmacokinetic Methods

Population pharmacokinetic methods are a population-based evaluation of
measurements of systemic drug concentrations, usually two or more per
patient under steady state conditions, from all, or a defined subset of,
patients who participate in clinical trials.
Pharmacokinetic, Pharmacodynamic, and Dose Response

Considerations
Evaluation of the pharmacokinetics and pharmacodynamics, and their

comparability, in the three major racial groups most relevant to the ICH
regions (Asian, Black, and Caucasian) is critical to the registration of
medicines in the ICH regions. Basic pharmacokinetic evaluation should
characterize absorption, distribution, metabolism, excretion (ADME), and
where appropriate, food-drug and drug-drug interactions. Adequate
pharmacokinetic comparison between populations of different regions
allows rational consideration of what kinds of further pharmacodynamic
and clinical studies (bridging studies) are needed for the new region. In
contrast to the pharmacokinetics of a medication, where differences
between populations may be attributed primarily to intrinsic ethnic factors
and are readily identified, the pharmacodynamic response (clinical
effectiveness, safety, and dose-response) may be influenced by both intrinsic
and extrinsic ethnic factors and this may be difficult to identify except by
conducting clinical studies in the new region.
In general, dose-response (or concentration-response) should be
evaluated for both pharmacologic effect (where one is considered pertinent)
and clinical endpoints in a new foreign region. The pharmacologic effect,
including dose-response, may also be evaluated in the foreign region in a
population representative of the new region.
Depending on the situation, data on clinical efficacy and doseresponse in
the new region may or may not be needed, e.g., if the drug class is familiar
and the pharmacologic effect is closely linked to clinical effectiveness and
dose-response, the foreign pharmacodynamic data may be a sufficient basis
for approval and clinical endpoint and dose-response data may not be
needed in the new region. The pharmacodynamic evaluation, and possible
clinical evaluation (including dose-response), is important because of the
possibility that the response curve may be shifted in a new population.

62 Mehta and Hunt
Examples of this are well documented, e.g., the decreased response in blood
pressure of blacks to angiotensin-converting enzyme inhibitors.
E11: “Clinical Investigations of Medicinal Products in the

Pediatric Population” Guideline (2000)
The sections of this guideline that outline the clinical pharmacology
information are:
Types of Studies
When a medicinal product is to be used in the pediatric population for the
same indication(s) as those studied and approved in adults, the disease
process is similar in adults and pediatric patients, and the outcome of
therapy is likely to be comparable, therefore extrapolation from adult
efficacy data may be appropriate. In such cases, pharmacokinetic studies in
all the age ranges of pediatric patients likely to receive the medicinal
product, together with safety studies, may provide adequate information for
use by allowing selection of pediatric doses that will produce blood levels
similar to those observed in adults. If this approach is taken, adult
pharmacokinetic data should be available to plan the pediatric studies.
When a medicinal product is to be used in younger pediatric patients for
the same indication(s) as those studied in older pediatric patients, the disease
process is similar, and the outcome of therapy is likely to be comparable,
therefore extrapolation of efficacy from older to younger pediatric patients
may be possible. In such cases, pharmacokinetic studies in the relevant age
groups of pediatric patients likely to receive the medicinal product, together
with safety studies, may be sufficient to provide adequate information for
pediatric use.
An approach based on pharmacokinetics is likely to be insufficient for
medicinal products where blood levels are known or expected not to
correspond with efficacy, or where there is concern that the
concentrationresponse relationship may differ between the adult and
pediatric populations. In such cases, studies of the clinical or the
pharmacological effect of the medicinal product would usually be
expected.
Where the comparability of the disease course or outcome of therapy in
pediatric patients is expected to be similar to adults, but the appropriate
blood levels are not clear, it may be possible to use measurements of a
pharmacodynamic effect related to clinical effectiveness to confirm the
expectations of effectiveness and to define the dose and concentration
needed to attain that pharmacodynamic effect. Such studies could provide
increased confidence that achieving a given exposure to the medicinal
product in pediatric patients would result in the desired therapeutic

outcomes. Thus, a PK/PD approach combined with safety and other

relevant studies could avoid the need for clinical efficacy studies.
In other situations where a pharmacokinetic approach is not applicable,
such as for topically active products, extrapolation of efficacy from one
patient population to another may be based on studies that include
pharmacodynamic endpoints and/or appropriate alternative assessments.
Local tolerability studies may be needed. It may be important to determine
blood levels and systemic effects to assess safety.
Pharmacokinetics
Pharmacokinetic studies generally should be performed to support
formulation development and determine pharmacokinetic parameters in
different age groups to support dosing recommendations. Relative
bioavailability comparisons of pediatric formulations with the adult oral
formulation typically should be done in adults. Definitive pharmacokinetic
studies for dose selection across the age ranges of pediatric patients in whom
the medicinal product is likely to be used should be conducted in the
pediatric population.
For medicinal products that exhibit linear pharmacokinetics in adults,
single-dose pharmacokinetic studies in the pediatric population may
provide sufficient information for dosage selection. This can be
corroborated, if indicated, by sparse sampling in multidose clinical studies.
Any nonlinearity in absorption, distribution, and elimination in adults and
any difference in duration of effect between single and repeated dosing in
adults would suggest the need for steady state studies in the pediatric
population. All these approaches are facilitated by knowledge of adult
pharmacokinetic parameters. Knowing the pathways of clearance (renal
and metabolic) of the medicinal product and understanding the age-related
changes of those processes will often be helpful in planning pediatric studies.
M4: “The Common Technical Document for the Registration

of Pharmaceuticals for Human Use. EFFICACY. Module 2:
Clinical Overview and Clinical Summary. Module 5: Clinical
Study Reports” (Step 4; 2000)
This is a very comprehensive guideline that identifies all important aspects
of clinical pharmacology and biopharmaceutic considerations and provides
details on format and content of related requirements. In view of the
authors, this is a comprehensive update of the United States guideline issued
in 1987 and is a must-read.
As stated in the title, module 2 in this guideline goes over the organization
and content of the clinical overview and the clinical summary sections.

64 Mehta and Hunt
Following this, module 5 provides organization of clinical study reports and

related information. These reports are broken down into seven different
categories: Biopharmaceutics Studies; Studies Pertinent to PK Using Human
Biomaterials; Human PK Studies; Human PD Studies; Efficacy and Safety
Studies; Postmarketing Experience; Case Report Forms; and Individual
Patient Listings. The first four of these report types form the basis for
clinical pharmacology and biopharmaceutics information required in an
application and are covered in detail below:
Biopharmaceutic Studies
This guideline states that bioavailability studies evaluate the rate and extent
of release of the active substance from the medicinal product. Comparative
BA or BE studies may use PK, PD, clinical, or in vitro dissolution endpoints,
and may be either single dose or multiple dose. Types of BA studies
identified are (i) studies comparing the release and systemic availability of a
drug substance from a solid oral dosage form to the systemic availability of
the drug substance given intravenously or as an oral liquid dosage form, (ii)
dosage form proportionality studies, and (iii) food-effect studies. Next set of
studies identified are comparative BA and BE studies, and these are studies
that compare the rate and extent of release of the drug substance from
similar drug products (e.g., tablet to tablet, tablet to capsule). Comparative
BA or BE studies may include comparisons between (i) the drug product
used in clinical studies supporting effectiveness and the to-be-marketed drug
product, (ii) the drug product used in clinical studies supporting
effectiveness and the drug product used in stability batches, and (iii) similar
drug products from different manufacturers. The final type of studies
identified are In Vitro—In Vivo Correlation studies, i.e., in vitro dissolution
studies that provide BA information, including studies used in seeking to
correlate in vitro data with in vivo performance.
Studies Pertinent to Pharmacokinetics Using Human Biomaterials

The guideline defines human biomaterials as proteins, cells, tissues, and
related materials derived from human sources, which are used in vitro or ex
vivo to assess PK properties of drug substances. The types of studies
identified are plasma protein binding studies, and hepatic metabolism and
drug interaction studies. Examples include cultured human colonic cells that
are used to assess permeability through biological membranes and transport
processes, and human albumin that is used to assess plasma protein binding.
Of particular importance is the use of human biomaterials such as
hepatocytes and/or hepatic microsomes to study metabolic pathways and to
assess drug-drug interactions with these pathways.

Human Pharmacokinetic Studies

According to the guideline, assessment of the PK of a drug in healthy
subjects and/or patients is considered critical to designing dosing strategies
and titration steps, to anticipating the effects of concomitant drug use, and
to interpreting observed pharmacodynamic differences. These assessments
should provide a description of the body’s handling of a drug over time,
focusing on maximum plasma concentrations (peak exposure), area-
undercurve (total exposure), clearance, and accumulation of the parent drug
and its metabolite(s), in particular those that have pharmacological activity.
The PK studies are generally designed to (i) measure plasma drug and
metabolite concentrations over time, (ii) measure drug and metabolite
concentrations in urine or feces when useful or necessary, and/or (iii)
measure drug and metabolite binding to protein or red blood cells.
On occasion, PK studies may include measurement of drug distribution
into other body tissues, body organs, or fluids (e.g., synovial fluid or
cerebrospinal fluid). These studies should characterize the drug’s PK and
provide information about the absorption, distribution, metabolism, and
excretion of a drug and any active metabolites in healthy subjects and/or
patients. Studies of mass balance and changes in PK related to dose (e.g.,
determination of dose proportionality) or time (e.g., due to enzyme
induction or formation of antibodies) are of particular interest. Additional
studies can also assess differences in systemic exposure as a result of changes
in PK due to intrinsic (e.g., age, gender, racial, weight, height, disease,
genetic polymorphism, and organ dysfunction) and extrinsic (e.g., drug-
drug interactions, diet, smoking, and alcohol use) factors. In addition to
standard multiple-sample PK studies, population PK analyses based on
sparse sampling during clinical studies can also address questions about the
contributions of intrinsic and extrinsic factors to the variability in the
dosePK-response relationship. Thus, the guideline identifies the following
types of studies as Human PK studies: Healthy subject PK and initial
tolerability; Patient PK and initial tolerability; Intrinsic factor PK; Extrinsic
factor PK; and Population PK.
Human Pharmacodynamic Studies

The guideline identifies these as (i) studies of pharmacologic properties
known or thought to be related to the desired clinical effects (biomarkers),
(ii) short-term studies of the main clinical effect, and (iii) PD studies of other
properties not related to the desired clinical effect. Because a quantitative
relationship of these pharmacological effects to dose and/or plasma drug
and metabolite concentrations is usually of interest, PD information is
frequently collected in dose response studies or together with drug

66 Mehta and Hunt
concentration information in PK studies (concentration-response or PK/PD

studies). The guideline states that dose-finding, PD and/or PK-PD studies
can be conducted in healthy subjects and/or patients, and can also be
incorporated into the studies that evaluate safety and efficacy in a clinical
indication. In some cases, the short-term PD, dose-finding, and/or PK-PD
information found in pharmacodynamic studies conducted in patients will
provide data that contribute to assessment of efficacy, either because they
show an effect on an acceptable surrogate marker (e.g., blood pressure) or
on a clinical benefit endpoint (e.g., pain relief). Thus the studies identified
here are healthy subject PD and PK/PD studies plus patient PD and PK/PD
studies.
The reader must note that the guideline clearly states that when these PD
studies are part of the efficacy or safety demonstration, they are considered
clinical efficacy and safety studies that should be included in Section 5.
Similarly, studies whose primary objective is to establish efficacy or to
accumulate safety should be included in Section 5.
Section 5 is beyond the scope of this chapter.
GLOSSARY
Bioavailability. The rate and extent to which the active ingredient or active
moiety is absorbed from a drug product and becomes available at the site of
action. For drug products that are not intended to be absorbed into the
bloodstream, bioavailability may be assessed by measurements intended to
reflect the rate and extent to which the active ingredient or active moiety
becomes available to the site of action.
Bioeqivalence. The absence of a significant difference in the rate and extent

to which the active ingredient or active moiety in pharmaceutical
equivalents or pharmaceutical alternatives becomes available at the site of
drug action when administered at the same molar dose under similar
conditions in an appropriately designed study. Where there is an intentional
difference in rate (e.g., in certain controlled release dosage forms), certain
pharmaceutical equivalents or alternatives may be considered bioequivalent
if there is no significant difference in the extent to which the active
ingredient or moiety from each product becomes available at the site of drug
action. This applies only if the difference in the rate at which the active
ingredient or moiety becomes available at the site of drug action is
intentional, is reflected in the proposed labeling, is not essential to the
attainment of effective body drug concentrations on chronic use, and is
considered medically insignificant for the drug.

Drug. Means (i) articles recognized in the official United States Pharmacopoeia,
official Homoeopathic Pharmacopoeia of the United States, or official
National Formulary, or any supplement to any of them; and (ii) articles
intended for use in the diagnosis, cure, mitigation, treatment, or prevention
of disease in man or other animals; and (iii) articles (other than food)
intended to affect the structure or any function of the body of man or other
animals; and (iv) articles intended for use as a component of any article
specified in clause (i), (ii), or (iii); but does not include devices or their
components, parts, or accessories.
Drug Product. A finished dosage form, e.g., tablet, capsule, or solution, that
contains the active drug ingredient, generally, but not necessarily, in
association with the inactive ingredients.
Extended Release. Extended release products are formulated to make the
drug available over an extended period after ingestion. This allows a
reduction in dosing frequency compared to a drug presented as a
conventional dosage form (e.g., as a solution or an immediate release dosage
form).
Immediate Release. Allows the drug to dissolve in the gastrointestinal

contents, with no intention of delaying or prolonging the dissolution or
absorption of the drug.
Interstate Commerce. Means (i) commerce between any State or Territory

and any place outside thereof, and (ii) commerce within the District of
Columbia or within any other Territory not organized with a legislative
body.
Labeling. All labels and other written, printed, or graphic matter (i) upon
any article or any of its containers or wrappers, or (ii) accompanying such
article.
Modified Release Dosage Forms. Dosage forms whose drug-release

characteristics of time course and/or location are chosen to accomplish
therapeutic or convenience objectives not offered by conventional dosage
forms such as a solution or an immediate release dosage form. Modified
release solid oral dosage forms include both delayed and extended release
drug products.
Pharmaceutical Alternatives. Drug products that contain the identical

therapeutic moiety, or its precursor, but not necessarily in the same amount
or dosage form or as the same salt or ester. Each such drug product
individually meets either the identical or its own respective compendial or
other applicable standard of identity, strength, quality, and purity, including

68 Mehta and Hunt
potency and, where applicable, content uniformity, disintegration times,

and/or dissolution rates.
Pharmaceutical Equivalents. Drug products that contain identical amounts
of the identical active drug ingredient, i.e., the same salt or ester of the same
therapeutic moiety, in identical dosage forms, but not necessarily containing
the same inactive ingredients and that meet the identical compendial or
other applicable standards of identity, strength, quality, and purity,
including potency and, where applicable, content uniformity, disintegration
times and/or dissolution rates.
ACKNOWLEDGMENT
The authors thank Mr. Donald Hare for his useful suggestions and input.
NOTES
1. The text of the Federal Food, Drug, and Cosmetic Act, as amended, can be
found codified in the United States Code (USC) under Title 21 (Food and Drugs).
Example, FDCA Section 505 for New Drugs can also be found in Section 355 of
Title 21 of USC (21 USC 355).
2. As a result of the disaster where it was discovered that the drug thalidomide
caused deformities in newborn children, the Kefauver-Harris Amendments
were added to the FDCA in 1962. These amendments covered or required
that (i) efficacy in addition to safety be demonstrated for a product, (ii) there
be good manufacturing practices (GMPs) for which products could be removed
from the market if not manufactured in conformity with current good
manufacturing practices (CGMPs) to ensure product quality, (iii) there be
implementation of investigational new drug applications (INDs), and (iv)
prescription drug advertising be put under FDA supervision while advertising
for over-the-counter (OTC) products would remain with the Federal Trade
Commission (FTC).
3. It is noted that all products that are approved via 505(b)(1) or 505(b)(2)
applications or as supplements to NDAs, if appropriate, are also included in the
Orange Book and are coded as appropriate among the different codes that are
allowed.
4. Before a rule or regulation is codified in the CFR, it is published as a proposed
rule or regulation in the FR for which public comment is requested and after
which it is finalized in a subsequent FR publication with modifications if needed.
In the CFR, relevant FR publications are usually referenced. The FR and CFR
can be accessed via the internet at http://www.access.gpo.gov/su_docs/
index.html.
5. Before a guidance is finalized, it is published as a draft in the FR in order to

obtain public comment. The finalized guidance is published in a subsequent FR

notice.
6. There are five steps in the ICH process of guideline development and issuance
which are Consensus Building (Step 1), Start of Regulatory Action (Step 2),
Regulatory Consultation (Step 3), Adoption of a Tripartite Harmonized Text
(step 4), and Implementation (Step 5).
REFERENCES
1. Federal Food, Drug and Cosmetic Act, as Amended, Supt. of Documents, U.S.
Government Printing Office: Washington, DC, 2001.
2. Approved Drug Products with Therapeutic Equivalence Evaluations, Supt. of
Documents, U.S. Government Printing Office: Washington, DC, 2001.
3. Federal Register, Supt. of Documents, U.S. Printing Office: Washington, DC.
4. Code of Federal Regulations, Title 21, Supt. of Documents, U.S. Government
Printing Office: Washington, DC, 2001.
5. Drug Bioequivalence: A Report of the Office of Technology Assessment Drug
Bioequivalence Study Panel, Supt. of Documents, U.S. Government Printing
Office: Washington, DC, 1974.

4
New Drug Application Content and
Review Process for Clinical
Pharmacology and Biopharmaceutics
Chandrahas Sahajwalla, Veneeta Tandon,

and Vanitha J.Sekar*
INTRODUCTION
The regulation and control of new drugs in the United States has been based
on the new drug application (NDA) that is evaluated by the U.S. Food and
Drug Administration (FDA). The data gathered in preclinical studies and
human clinical trials as an investigational new drug (IND) during the drug
development process become part of the NDA. The goal of the drug
development process is to provide sufficient information to the FDA in the
NDA to evaluate the efficacy and safety of the new drug as well as
recommendations to adjust the dose in special circumstances. The drug
development process for new drugs has evolved over the years especially in the
field of Clinical Pharmacology and Biopharmaceutics. In response to the
* Current affiliation: Aventis Pharmaceuticals, Bridgewater, New Jersey, U.S.A.
71
72 Sahajwalla et al.
evolving technology, advancement of knowledge in the field, and to ascertain

consistency and quality of the data available during the development process,
the US FDA, including, office of clinical pharmacology and biopharmaceutics
(OCPB) has issued several regulatory guidance documents. Office of clinical
pharmacology and biopharmaceutics has several guidances in the public
domain that are available to drug companies (often referred to as sponsors)
which provide recommendations in the areas of clinical pharmacology/
biopharmaceutics such as exposure-response assessments, design and conduct
of population pharmacokinetic studies, in vitro and in vivo drug metabolism
and drug interactions, dissolution testing requirements for immediate and
extended release dosage forms, design and conduct of bioavailability,
bioequivalence and food-effect studies, and studies in patients with renal and
hepatic impairment. This chapter integrates the information from available
OCPB and other FDA-issued guidances that aid in the drug development
process, and also provides insight into some of the issues that should be
considered from a regulatory perspective regarding the Clinical
Pharmacology and Biopharmaceutics aspects of drug development. It should
be noted that some of the guidances are published as a draft and reflect
current scientific understanding and thinking of the FDA scientist.
The sponsors now have option submitting new drug application in NDA
format or Common Technical Document (CTD) format. Common technical
document format is a format in which clinical, pharmacology/ toxicology
and manufacturing data can be submitted to obtain marketing
authorization for new drugs in the United States, European Union, and
Japan. It should however be noted that CTD and NDA do not differ in the
content of the information but mainly the format in which data should be
provided.
This chapter provides an insight into the review process by the Clinical
Pharmacology and Biopharmaceutics staff.
STAGES IN DRUG DEVELOPMENT AND REGULATORY

PROCESS
Once the sponsor has identified a lead compound, traditionally, the drug
development process follows a plan. Most pharmaceutical companies have
a drug development plan that is unique to their company based on their own
experiences. In general all pharmaceutical companies proceed with
development to answer several questions about the drug, i.e., is the drug
safe, up to what dose or exposure it is safe, how should the dose be adjusted
in certain specific populations or when co-administered with other drugs to
have optimized formulation for delivery of the drug.

When a compound has been identified, a Pre-IND (IND-investigational

new drug) meeting is occasionally requested with the FDA by sponsors.
Sponsor may be a pharmaceutical company or individual investigators.
Prior to the meeting, the sponsor usually submits a Pre-IND package. The
Pre-IND package may include summary of preclinical data and a concept
sheet of a study protocol in order to obtain scientific input from the FDA
reviewers regarding the initial IND. The FDA review team consists of a
Medical Officer, Clinical Pharmacologist and Pharmacokineticist, Chemist,
Pharmacologist/Toxicologist Statistician, and a Microbiologist (depending
on the proposed indication). Input requested by the sponsor before the filing
of the initial IND usually involves questions regarding appropriate dose
and/or dosing regimen selection, safety parameters to be assessed, sampling
times (pharmacokinetics and safety), etc., for the “first time in humans”
study. Generally, the first study conducted in human volunteers is a clinical
pharmacology study to evaluate the safety and pharmacokinetics/
pharmacodynamics of the drug in healthy volunteers or, in some cases,
patients. Prior to conducting this first-time-in-humans study, the FDA
requires the sponsor to have conducted adequate preclinical studies to
support such a study. The sponsor may also request FDA input regarding
the development plan for their compound, generally if human data on the
drug is available from studies conducted outside the USA. In this case, the
OCPB reviewer would review the sponsor’s plan and provide additional
suggestions, whenever necessary. Examples of OCPB input at the Pre-IND
stage regarding overall drug development include formulation development
plans, dissolution method development, exploring mechanisms of action,
design and conduct of in vitro metabolism studies, clinical pharmacology
study designs, identifying potentially useful biomarkers, proof of concept
and doseranging studies, exposure-response and/or population
pharmacokineticpharmacodynamic assessments, as well as design and dose
selection plans for Phase 3 studies. Depending on the complexity of the Pre-
IND, the Agency would respond either via a letter or a meeting may be set
up with the sponsor.
Protocols for all studies conducted in human volunteers in the United
States or that would become part of the NDA have to be submitted to the
FDA. Once an IND has been filed FDA assigns a number to the IND.
Subsequent study protocols, study reports or sponsor’s correspondences
have to refer to the IND number.
Once the sponsor has submitted an IND to the FDA, FDA has 30 days to
review the submitted protocol for human study. During this review, if there
are any concerns about the safety of the subjects to be enrolled in the study,
FDA would call the sponsor and place the protocol on clinical hold until the
concerns identified by the FDA reviewers are satisfactorily addressed. The
IND review process is shown schematically in Fig. 1.

74
Sahajwalla et al.
FIGURE 1 The IND review process, http://www.fda.gov/cder.
There is keen interest on the part of the pharmaceutical companies to be

involved in screening INDs (at the time of the initial IND submission) in
which several drugs are screened at the same time and one of the
compounds is identified for further development. Further details of this
approach can be found in manual of policy and procedures (MAPP) on the
FDA website [1].
The drug development stages are not rigid, that is, several phases of early
drug development (traditionally called Phase 1 and 2 studies) are generally
on going simultaneously. Typically, Phase 1 studies are in healthy
volunteers, Phase 2 are studies in small numbers of patients, and Phase 3 are
larger clinical trials with adequate number of subjects to determine safety
and efficacy of the drug. Phase 1 studies typically include studies related to
formulation development, assessment of metabolic pathways, assessment of
effects of extrinsic and intrinsic factors such as age, gender, disease, other
drugs and food, and assessment of PK—PD. Phase 2 studies are typically
dose-ranging and proof of concept studies in a small number of patients
who comprise the target population (traditionally called Phase 2A).
Assessment of PK-PD is also performed in these studies to help provide an
understanding of the doses and dose regimens to be further studied. These
studies provide the sponsor as well as the regulatory agencies with the type
of knowledge about the drug that is needed to design appropriate
confirmatory or definitive large clinical trials in the target patient
population (traditionally known as Phase 3 trials). Generally, the FDA needs
two positive adequately well controlled Phase 3 trials that support the safety
and efficacy of the drug in the target population prior to approval for
marketing in the U.S. The overall drug development stages are shown
schematically in Fig. 2.
Prior to the start of definitive efficacy or Phase 3 trials, the sponsor
usually requests to meet with the FDA at an End-of-Phase 2 meeting. At this
meeting, the sponsor discusses with the Agency the information that has
been learned about the clinical pharmacology and the limited information
obtained in patients about the safety and efficacy of the drug. End-of-Phase
2 meeting discussions with the FDA usually revolve around the decision as
to whether the sponsor should proceed to conduct the larger Phase 3 trials
and, if so, the appropriate study design for these larger Phase 3 studies.
Clinical trial simulations using the in vitro and in vivo data collected from
the early phases of development may also aid in optimal design of the Phase
3 trials. The sponsor can request a special protocol assessment [1] for
evaluating issues related to the adequacy (e.g., design, conduct, analysis) of
certain proposed studies associated with the development of their drug
products. Three types of protocols are eligible for this special protocol
assessment: (1) animal carcinogenicity protocols, (2) final product stability
protocols, and (3) clinical protocols of Phase 3 trails whose data will form

FIGURE 2 Stages in drug development and regulatory process. http://www.

fda.gov/cder.
the primary basis for an efficacy claim (if the trials had been discussed at an
End-of-Phase 2/pre-Phase 3 meeting or if the review division is aware of the
developmental context in which the protocol is being reviewed). The FDA
has 45 days to review the protocol and provide scientific/regulatory
comments to the sponsor as needed [2]. The guidance recommends that a
sponsor submit a protocol intended for special protocol assessment to the
Agency at least 90 days prior to anticipated commencement of the study.
The protocol should be complete and sufficient time should be allowed to
discuss and resolve any issues before the study begins. Special protocol
assessments are not to be provided after a study has begun.
There is also a keen interest on the part of the sponsors and the FDA to
have a pre-Phase 2 meeting (Phase 2A meeting; i.e., prior to starting the
pivotal Phase 2 study in a small set of patients). During this meeting,
information available on preclinical studies and Phase 1 studies conducted
up to that time can be integrated to assess and discuss Phase 2 protocols.
These meetings could provide great opportunity to discuss dosing rationale
for the Phase 2 trials, evaluation of appropriate biomarkers, and assessment
of exposure-response relationships. There is great interest in these early
interactions between the sponsor and the FDA because resources can be
used more efficiently and effectively by early communications. There is great

opportunity for the sponsor and FDA to identify any limitations in the drug
development plan early on, so that all relevant information is available at
the time NDA/CTD is submitted to the FDA. These meetings have potential
to reduce number of review cycles that some times result, and to produce a
better drug product label.
Data and information from all studies conducted during the IND phase
are summarized and submitted in one package, i.e., NDA. Prior to
submission of the NDA, generally the sponsor requests the FDA for a face-
to-face Pre-NDA meeting (usually a few months prior to the submission of
the NDA). Issues discussed during this meeting include the content and
format of the different sections of the NDA that would be considered
“fileable,” including issues related to electronic submission of the NDA. At
this meeting, assessment is also made if any critical piece essential for
regulatory decision-making is missing. The FDA has issued a guidance to the
industry on the format and content of electronic submissions that are made
to the Agency and are available on the FDA Website.
Once an NDA is submitted to the FDA, the agency assigns an NDA
number to the drug. Since not all drugs being investigated as IND become a
successful candidate for marketing, it should be noted that NDA number is
a different number than an IND number. Once an NDA has been submitted,
all correspondence for that NDA should reference that NDA number. FDA
has 60 days to file that submitted NDA, or FDA could refuse to file an NDA
due to format and content issues or absence of critical piece(s) of
information/data needed for the FDA to make a decision on the
approvability of the NDA.
Under the Prescription Drug User Fee Act of 1992 (PDUFA), the FDA
has defined timeframes applicable to drug application reviews. The FDA
usually takes 6 to 10 months from the date of submission of the NDA to
make a decision of the acceptability of the application, often referred to as
NDA action. This time frame depends on the type of NDA submitted. The
FDA gives a priority designation for a product that if approved would be a
significant improvement compared to marketed products in the treatment,
diagnosis, or prevention of a disease. Evidence of increased effectiveness,
elimination, or reduction of treatment related drug reactions, safety, and
effectiveness in a new subpopulation, or enhanced patient compliance can
demonstrate improvement. All applications not qualifying as priority are
classified as standard applications. Priority applications are reviewed
within six months, where as standard applications have a 10-month
review clock. A decision regarding the assignment of a standard or a
priority rating to the application is made before the 60 day filing of
the NDA.
There are certain types of drug approval processes that facilitate the
development and expedite the review of the new drugs that are intended to

treat serious life threatening conditions and to demonstrate the potential

as treatment for an unmet medical need. Some of these programs are the
accelerated drug approval/fast track programs or rolling submissions. The
accelerated drug approval program (Subpart H) is a highly specialized
mechanism for speeding the development/review of drugs that promise
significant benefit over existing therapy for serious or life-threatening
illnesses like AIDS, cancer, Parkinson’s disease etc., and for a condition for
which no therapy exists. This program involves the modification of the
criteria on which the approval is based on. It allows for approval to be
based on a surrogate endpoint or an effect on a clinical end point other
than survival or irreversible morbidity. Under such circumstances, the
program may require appropriate post approval studies to validate the
surrogate endpoint or otherwise confirm the effect on a valid clinical
endpoint.
When certain sections of an application are accepted by the Agency prior
to the receipt of the complete application, the submissions are referred to as
rolling NDA submissions (i.e., pre-submission of pharm-tox reports, clinical
study reports, and even data summaries and listings from the first of two or
more pivotal trials). Sponsors of designated fast track products can request
this type of submission by submitting certain completed portions of an NDA
prior to submitting the other sections of the application. In such cases the
sponsor is required to provide a schedule for submitting the information
necessary to make the NDA submission complete. Further details of these
programs can be found under Regulatory Guidance and Mapp (Manual of
Policy and Procedure) on the FDA website [1, 3].
Sometimes there is a need for either an Advisory Committee Meeting or a
face-to-face meeting with the sponsor to discuss issues that arise during the
NDA review process. Once the NDA is submitted, pivotal study sites are
identified and inspected for good clinical practices (GCP) and good
laboratory practices (GLP) compliance by the Office of Compliance. An
NDA action is taken after obtaining results from the inspection of the study
site. The action could result in the approval or non-approval of an NDA, or
in an approvable NDA. An approvable NDA implies that the information
that has been reviewed by the FDA appears to be an acceptable data;
however, some additional information is needed to approve the product for
marketing in the United States. This could involve collection of additional
data, data re-analysis or negotiation of labeling language. The overall NDA
review process is shown schematically in Fig. 3.
Table 1 summarizes the type of studies that are typically part of the
clinical pharmacology and biopharmaceutics plan for a new drug, and Table
2 gives an example of how all of the clinical pharmacology and
biopharmaceutics information can be summarized concisely. Readers are

Clinical Pharmacology and Biopharmaceutics
79
FIGURE 3 NDA review process, http://www.fda.gov/cder.
TABLE 1 General list of Studies Submitted to Support the Clinical Pharmacology

and Biopharmaceutics Portion of the NDA

also encouraged to refer to the FDA website and the ICH Common
Technical Document that provides information on what information an
new drug application should contain.
CLINICAL PHARMACOLOGY CONSIDERATIONS IN NEW

DRUG DEVELOPMENT
In a new drug application, the OCPB reviewer is looking for data and
analyses that provide a rational justification for the selected dose/dosing
regimen as well as the sponsor’s attempt to “individualize” doses in certain
populations and/or scenarios, e.g., in pediatrics, in elderly, in renal/hepatic
impairment, and in presence of concomitant medications. The sponsor
usually generates this information in the IND stage of the regulatory
process. The reader is also encouraged to read the article that describes the
question-based review approach that the Office of Clinical Pharmacology
and Biopharmaceutics follows [4]. The chapters presented in this book
provide a general approach to drug development.
There may be some classes of drugs with certain characteristics (e.g.,
chirality), formulation (e.g., liposomes) or certain indications (e.g.,
biologicals) which may need additional consideration in their evaluation.
Some of these cases are discussed in various chapters of this book.
BIOPHARMACEUTICS CONSIDERATIONS IN NEW DRUG

DEVELOPMENT
Biopharmaceutics is a comprehensive term denoting the study of the

influence of pharmaceutical formulation variables on the performance of
the drug in vivo [5]. In a new drug application, the OCPB reviewer generally
looks for the pH solubility profile, pKa of the drug substance, drug
permeability or octanol/water partition coefficient measurement which may
be useful in selecting the dissolution methodology and specifications.
Dissolution of the drug under physiological conditions is one of the
factors assessing drug absorption after oral administration. Dissolution
testing is required for all solid oral dosage forms in which absorption of the
drug is necessary for the product to exert the desired therapeutic effect. In
addition to predicting in vivo performance of the dosage units, dissolution
tests help in assuring drug product quality from batch to batch and may also
be a guide in the development of new formulations. The dissolution
specifications set forth also ensure the drug product’s sameness under
scaleup and postapproval changes. Dissolution data also provides for
assessing the waiver of a bioequivalence study. For NDAs the dissolution

82
TABLE 2 Summary of Clinical Pharmacology and Biopharmaceutics Characteristic of the Drug
Sahajwalla et al.
specifications are based on acceptable clinical, pivotal bioavailability and/or

bioequivalence batches.
Biopharmaceutics issues depend on the route of administration as well as
the kind of dosage forms (oral versus other routes of administration,
immediate release dosage form, and modified release dosage forms). Some
of these issues have been covered in the various chapters of this book.
The final formulation the sponsor wishes to market may not always be
the one that has been used during the drug development. These formulation
changes may be necessary due to variety of reasons ranging from aesthetic to
overall improvement in formulation performance or to accommodate
manufacturing convenience. It is essential to know that the to-be-marketed
formulation will perform in the same way as the clinical trial formulation
performed in the pivotal clinical studies. For an NDA, bioequi valence
studies provide a link between the pivotal and early clinical trial
formulation, a link between the formulations used in the pivotal clinical
trial, and the to-be-marketed formulation or any other comparisons as
appropriate. Bioequivalence studies provide information on the product
quality and performance, when there are changes in components,
composition and method of manufacture after approval of the drug
product. The FDA has provided Guidance for the industry, such as BA/BE
guidance [6], SUPAC-IR [7], and SUP AC-MR [8], to determine when the
changes in the components and composition and/or method of manufacture
of the drug product suggest a need to perform further in vitro/in vivo
studies. Although, SUP AC stands for Scale-up and Post Approval Changes
to the formulation, the same principals outlined in these guidances are
utilized at the preapproval stage of the drug to determine the level of data
needed for bio waivers.
PRODUCT LABEL
One of the most important products of the drug development is the drug
product labeling. Since this is the document that will be utilized by the
prescribing Physicians to appropriately dose the patients, great care is taken
by the FDA and Industry Scientist to provide accurate information in a clear
and concise way in the product labeling. Labeling guides the prescriber,
based on data obtained from clinical trials, in optimizing the dose and
dosage regimen for all populations and outlines the adverse events which
were experienced by patients in the clinical trials etc. Labeling generally has
the following subheadings: Warnings, Description, Chemical Structure,
Clinical Pharmacology, Indication and Usage, Contraindications,
Precautions, Adverse Reactions, Overdosage, Dosage and Administration,
How Supplied, and Product Photos. In general, Clinical Pharmacology

sections describe the clinical studies conducted to obtain pharmacokinetic

data in healthy subjects, patients, special populations and drug-drug
interactions.
Precautions and contradictions will generally highlight data that would
require a caution or adjustment of dose. The dosage administration section
gives the approved dose and recommended dosage adjustments under
special circumstances. Presently, there is an initiative where a working group
at FDA is working on reforming the label so that important information for
the prescriber is highlighted in the beginning of the label.
SUMMARY
Drug development is a complex process that requires collaboration of

scientists with varying expertise. For any new drug being developed, teams
of scientists are responsible within an industry to develop the drug, and a
team of scientists at the FDA are responsible to review the IND and NDA
submitted to the FDA.
Involvement of FDA scientists generally starts with the submission of a
pre-IND meeting request by the sponsor. Although FDA scientists are
involved and interact with the sponsor during the entire drug development
process, some of the key interaction occurs when the sponsor submits an
IND, drug development plan, pre-Phase 2 meetings, End-of-Phase 2
meeting, pre-NDA meeting, and when the protocols are submitted during
the IND phase of development. For optimal drug development, FDA
encourages sponsor to have open communication and reviewers are
available to meet the industry scientists at any stage of drug development.
These meetings provide a forum for interactive exchange of scientific ideas.
To encourage and facilitate meeting between the industry and sponsor
scientists, a document describing process of arranging meetings has been
published as manual for policies and procedures for meetings and is
published on the FDA website [1].
For ease of understanding and getting an overview of the drug
development, it is important to summarize the assessment of new drug
application in one table. One example of such a table has been provided in
Table 2 in this chapter.
Once the FDA scientist has completed the review, the important part is to
convey the data in a clear way, so that the physicians can make informed
decision as to what is best for the patients. Readers are encouraged to look
at completed NDA reviews available on FDAs, Freedom of Information
(FOI) Website to gain insight into the regulatory issues that may arise during
reviews of NDAs.

In this chapter we have briefly covered the IND and NDA review process.
However, it is beyond the scope of this book to cover in detail several
regulatory considerations such as good clinical practices, good laboratory
processes, advisory committee meetings, orphan drugs, supple-mental
NDA, post approval changes, etc. Readers are referred to the FDA, ICH,
and other regulatory agency Websites to get additional information or
updates on scientific and regulatory issues related to new drug development.
REFERENCES
1. http://www.fda.gov/cder/guidance/index.html
2. Guidance for Industry: Special Protocol Assessment, Food and Drug Admini-
stration, May 2003.
3. Guidance for Industry: Fast Track Development Programs-Designation,
Development and Application review, Food and Drug Administration, September
1998.
4. Lesko, L.J.; Williams, R.L. The Question-Based Review: A Conceptual
Framework for Good Review Practices. Applied Clinical Practice 1999,8, 56–
62.
5. Rowland; Tozer. Clinical Pharmacokinetics. Concepts and Application, 3rd Ed.,
Williams and Wilkins, 1995.
6. Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally
Administered Drug Products—General Considerations, Food and Drug
Administration, October 2000.
7. Guidance for Industry: Immediate Release Solid Oral Dosage Forms Scale-Up
and Postapproval Changes: Chemistry, Manufacturing, and Controls, In-Vitro
Dissolution Testing, and In-Vivo Bioequivalence Documentation, Food and Drug
Administration, November 1995.
8. Guidance for Industry: SUPAC-MR: Modified Release Solid Oral Dosage Forms
Scale-Up and Postapproval Changes: Chemistry, Manufacturing, and Controls;
In Vitro Dissolution Testing and In Vivo Bioequivalence Documenta-tion, Food
and Drug Administration, October 1997.

5
In-vitro Drug Metabolism Studies During
Development of New Drugs
Anthony Y.H.Lu
Rutgers University
Piscataway, New Jersey, U.S.A.
Shiew-Mei Huang
INTRODUCTION
Since late 1980s, the drug discovery and development process has
undergone significant changes, particularly in the preclinical stage involving
drug candidate selection, drug metabolism and safety studies. These changes
are directly related to the scientific progress in research areas of
combinatorial chemistry, recombinant DNA technology, toxicology,
metabolism, and analytical instrumentation. The increasing availability of
tissues, cell cultures, and drug-metabolizing enzymes from human sources
has led to the increased use of in vitro studies to select the most desirable
drug candidates. Well executed in vitro studies can provide valuable
information regarding the metabolic fate of a new drug in humans, critical
87
88 Lu and Huang
factors contributing to the variability of pharmacokinetic parameters, and

the potential for drug-drug interactions. Consequently, in vitro study results
are now being routinely included in New Drug Applications (NDA) by the
sponsors.
What type of in vitro studies should be included in the NDA? How
should these studies be conducted? In this chapter, we describe some of the
commonly used in vitro techniques used to study drug metabolism during
drug development. However, as indicated in an FDA document on in vitro
drug metabolism studies [1], the assessment of drug metabolism in vitro is a
rapidly evolving area of drug development and regulation. Therefore, new
methods and additional studies will undoubtedly be added to this list. Since
one of the guiding principles in drug development is to generate data
utilizing up-to-date scientific technology and knowledge available in the
field, modification of currently used methods and approaches are expected
with time. The goal of early in vitro studies conducted at the preclinical
stage is to obtain optimal information to maximize the possibility of success
in developing a safe and effective drug for clinical use.
METHODS TO ASSESS DRUG-DRUG INTERACTION POTENTIAL
In vitro studies are useful for assessing the potential of metabolism-based

drug-drug interaction [2–4], a major concern for the effective and safe use of
therapeutic agents and a critical factor contributing to the recent
withdrawal of various drugs from the United States market [5–6]. Since
cytochrome P450 plays a key role in the metabolism of numerous important
drugs in clinical use, cytochrome P450-mediated drug-drug interactions
have attracted most attention, although the importance of transporter-
based drug-drug interactions has also been recognized in the last few years.
Central to the issue of metabolism-based drug-drug interactions is the
identification of the cytochrome P450(s) responsible for the metabolism of
the interacting drugs. Major activity alterations of the involving cytochrome
P450 species, due to either inhibition or induction, can result in potential,
significant pharmacokinetic changes of interacting drugs in humans.
As described in the following sections, various in vitro methods can be
used to assess the potential of drugs acting as inhibitors or inducers of
cytochrome P450. If the potential for interaction is great, in vivo studies in
human should be considered to evaluate the clinical significance of the in
vitro findings. The in vivo approaches include specific pharmacokinetic and
pharmacodynamic studies, population pharmacokinetic studies, and
clinical safety and efficacy studies [7–9]. In vivo animal studies have limited
values in predicting human drug-drug interactions, particularly if the results
in animals are negative. A single change in amino acid of the protein

In-vitro Drug Metabolism Studies 89
sequence can dramatically change the substrate specificity of cytochrome

P450 [10, 11]. In addition, various researchers have described species
differences in cytochrome P450 inhibition [12, 14] and induction [13].
Thus, cytochrome P450s in the same gene family in animals and human may
not respond to inhibitors and inducers in similar manners.
GENERAL APPROACHES
In vitro Methodologies
Most of the in vitro metabolism studies involve the use of tissues or drug-
metabolizing enzymes from the liver. The emphasis of metabolic research
has been on the liver, as it is considered the major organ for drug
metabolism, and that we know the most about the properties and
functions of liver drug-metabolizing enzymes, particularly cytochrome
P450. In addition, human liver tissues and human recombinant
cytochrome P450s are readily available. However, for some drugs,
nonhepatic tissues, such as the gastrointestinal mucosa, may play a vital
role in their metabolism. In these cases, in vitro metabolism studies
employing tissues from the kidneys, intestines, or skin may be valuable.
Similarly, although cytochrome P450s are the dominant enzymes for the
metabolism of most drugs, other drug-metabolizing enzymes are also
present in the liver and extrahepatic tissues. These non-cytochrome P450
enzymes are responsible for glucuronidation, sulfation, acetylation,
glutathione conjugation, and other enzymatic reactions. In vitro studies
using specific tissue fractions and cofactors are critical in characterizing
these metabolic reactions. In this chapter, unless specifically indicated, all
in vitro studies refer to cytochrome P450-mediated hepatic metabolism of
new drugs.
Many in vitro models are available to study hepatic drug metabolism,
ranging from the simplest recombinant enzymes to subcellular fractions,
hepatocytes, liver slices, to the more complicated isolated, perfused liver.
The degree of physiological relevance of these models decreases as one
changes from the whole organ to the recombinant enzymes. It is important
to select in vitro systems that are most suitable to achieve specific goals of
the study [2]. If the hepatic subcellular fractions are to be used for
metabolism studies, it is important to recognize the distribution of the
enzymes responsible for the metabolic events in various tissues and the
specific cofactors required for particular reactions.
One critical issue in conducting in vitro metabolism studies is the
appropriateness of drug concentrations that are used in these studies. Since
the drug concentration at the enzyme active site in the liver could not be

90 Lu and Huang
easily measured and the plasma drug concentration is generally unknown at

the time of in vitro metabolism study, it is often difficult to define the in vitro
drug concentration of physiological relevance.
Despite this uncertainty, it is the general rule not to use unrealistically
high drug concentrations (e.g., in the mM range) for in vitro metabolism
studies. Considering the assay sensitivity and the general plasma drug
concentrations in humans, drug concentrations in the low µM range
represent a good range to study for most of the in vitro metabolism studies.
A good practice is to use several drug concentrations (e.g., low, medium, and
high, spanning two to three orders of magnitudes) in these studies. This is
desirable particularly for drugs that undergo metabolism via two or more
pathways involving multiple enzymes (with different Km values). In this
case, both high and low affinity metabolic pathways can be studied.
With the advancement in analytical methodologies and knowledge of
human drug-metabolizing enzymes, the major metabolic pathways of a new
drug in humans can be readily established and metabolites can be isolated
from in vitro models. If the metabolites are found to be pharmacologically
active, sensitive and specific assays could be developed to assess the
pharmacokinetic profile of the metabolite(s) in subsequent clinical studies.
Animal toxicity studies are an important component of safety evaluation
of new drugs. Comparative animal and human metabolic profiles generated
in vitro can help the selection of appropriate animal models for toxicity
evaluation and may be useful in the interpretation or hypothesis-generating
of certain clinical findings.
The liver slices and hepatocyte suspensions from human and animal
species are suitable for metabolic profiling, since these systems contain all
the necessary enzymes and cofactors for metabolism [2]. Hepatic subcellular
fractions and recombinant drug-metabolizing enzymes can be used when
metabolic profiles are relatively simple and only one or two well-recognized
enzymes are involved in the biotransformation of the new drug. Because of
the known genetic polymorphism of many of the human drug-metabolizing
enzymes and the well-recognized large inter-individual variability in drug
metabolism, it is desirable to use liver tissues derived from more than one
individual (if possible) to generate metabolic profiles. In addition, as fresh
human livers are not always readily available, cryopreserved human
hepatocytes are now being increasingly used for drug metabolism studies
[3]. Cryopreserved human hepatocytes retain most, if not all, of the major
drug-metabolizing enzyme activities.
In vitro/In vivo Correlation

Although significant progress has been made in recent years in the
evaluation of drug-drug interaction potential based on in vitro data, a

complete understanding of the relationship between in vitro findings and in

vivo human results of metabolism-based drug-drug interaction studies is
still emerging. In some cases, excellent correlation of in vitro and in vivo
results has been demonstrated while in others, the in vitro and in vivo
correlation has been poor [15]. Because of the complexities of various
factors impacting both in vitro and in vivo drug-drug interactions, accurate
predictions of the extent of in vivo drug interactions from in vitro metabolic
studies will require continued efforts in obtaining additional high quality
correlation data to permit rational evaluation of new drugs. At the present
time, the feasibility of predicting in vivo drug interactions based on in vitro
metabolic data is still under rigorous debate. Some investigators believe that
a quantitative prediction of in vivo drug interaction is possible [16–18]
while others take the position that a qualitative prediction approach is more
feasible [19, 20]. In a recent commentary, Tucker et al. [21] used the
qualitative terms “low risk, medium risk, and high risk” to describe the
projection of AUC changes based on the [I]/Ki ratio, where the Ki values are
determined from in vitro studies.
Various factors contributing to the difficulty in predicting if a new
molecular entity (NME) is an inhibitor from in vitro data. Among them,
the unusual cytochrome P450 property and the large number of drug
substrates appear to be critical factors. In vitro drug-drug interaction
patterns (e.g., mutual inhibition, partial inhibition, activation, and lack of
reciprocal inhibition) for a given cytochrome P450, such as CYP3A4, are
often substrate-dependent. The Ki value of an inhibitor for a given
cytochrome P450 is dependent on the probe substrates, enzyme sources,
and experimental conditions such as protein concentration and incubation
time due to various degrees of inhibitor-protein binding, partition of
inhibitor to the lipid and aqueous layers, and inhibitor and substrate
depletion.
One of the challenges in predicting the extent of in vivo drug-drug
interaction from in vitro metabolism studies is the lack of information on
the inhibitor concentration in vivo in the active site of the enzyme or tissues.
Since the plasma inhibitor concentration may be the only known parameter,
both total inhibitor concentration and unbound inhibitor concentration
have been used for in vitro-in vivo correlation evaluation. Claims of good
correlation with either of the parameters have been reported for different
drugs. Other factors contributing to the lack of good in vitro-in vivo
correlation using either of the parameters may include the following: (1) the
inhibiting drug may also act as an inducer; (2) other parallel elimination
pathways and/or extrahepatic metabolism of the drug may decrease the
importance of the in vitro-assessed pathway; (3) modulation of an
important cellular transport mechanism by the inhibitor may change the
extent of in vivo drug-drug interaction, and (4) rapid elimination of

92 Lu and Huang
inhibitor in vivo by noncytochrome P450 pathways may decrease the extent

of in vivo drug-drug interaction.
Study Design Considerations

Cytochrome P450 Identification
Unequivocal identification of one or more specific cytochrome P450
enzymes responsible for the metabolism of new therapeutic agents is the
cornerstone of in vitro metabolism studies. This information is also critical
for the follow-up cytochrome P450 inhibition and induction studies in the
overall evaluation of in vitro drug-drug interactions. For all these studies,
the experimental conditions should be that the measured initial reaction
rates (in terms of product formation) are linear with respect to enzyme
concentration and incubation time. It is preferable to use low enzyme
concentration (e.g., below 0.5mg human liver microsomal protein per mL)
and short incubation time (less than 20 min) to minimize protein binding
and depletion of substrate and inhibitor (no more than 20% consumption,
preferably less than 10%). If the analytical sensitivity is not an issue, lower
enzyme concentration and shorter incubation time are highly desirable. In
case of a slow substrate turnover, higher enzyme concentration and longer
incubation time can be used as long as the initial metabolic rates are being
measured.
If the cytochrome P450-mediated metabolism represents a significant
clearance mechanism for the NME, cytochrome P450 reaction phenotyping
should be carried out, generally, with human liver microsomes and
recombinant cytochrome P450s using a combination of several basic
approaches [22]. The NME concentrations used are generally at or below
the Km values. Initial reaction rates are measured in the absence and the
presence of antibodies or chemical inhibitors, or with a panel of human liver
microsomes for correlation analysis with various cytochrome P450 probe
substrates. If there is an indication for the involvement of more than one
cytochrome P450 in the metabolism of the drug, several drug
concentrations (e.g., low, medium, and high-spanning two to three orders of
magnitude) should be used for inhibition studies.
Chemical Inhibitors and Inhibitory Antibodies. Specific and potent
inhibitors are valuable for cytochrome P450 reaction phenotyping. In this
respect, inhibitory antibodies (particularly monoclonal antibodies) with
demonstrated specificity and potency can be useful [23], as illustrated in a
recent paper by Granvil et al. [24]. These investigators described that the 4-
hydroxylation of debrisoquine, a well-recognized probe reaction of
CYP2D6, is mediated not only by CPY2D6 but also by human CYP1A1.
Whereas quinidine, a recognized selective inhibitor of CYP2D6, inhibits the

4-hydroxylation of debrisoquine by both CYP2D6 and human CYP1A1,

anti-CYP2D6 monoclonal anitbody inhibits specifically CYP2D6-
medicated reaction, and not CYP1A1-dependent metabolism. To date,
specific and potent monoclonal as well as polyclonal antibodies have not
been widely used by the pharmaceutical industry possibly due to their high
cost and limited availability from commercial sources.
A desirable antibody inhibition study can be conducted in two stages.
Initially, metabolism of a drug by pooled human liver microsomes is
examined in the presence of antibodies against all major human
cytochrome P450s at a single high concentration (known to give greater
than 80–95% inhibition with probe substrates) to determine which
antibodies significantly inhibit the metabolism. This study establishes that
one or more cytochrome P450 is involved in the metabolism of an NME.
In subsequent studies, the effect of those inhibitory antibodies on the
metabolism of the NME is studied in more detail using a series of antibody
concentrations. A well-designed study should show that metabolism is
inhibited strongly by the specific antibody in a concentration-dependent
manner at low antibody concentrations and then reaches maximum
inhibition at higher antibody concentrations [25] as illustrated in Fig. 1
(curves A and D). A steep inhibition slope indicates high potency of the
antibody against specific cytochrome P450. The extent of the maximum
inhibition indicates the extent (%) of the metabolism of the NME by this
particular cytochrome P450 enzyme. No meaningful conclusion can be
made regarding the role of a specific cytochrome P450 in the metabolism
of an NME when an antibody inhibition study showed a shallow
inhibition slope (an indication of low antibody potency) and failed to
demonstrate maximum inhibition (Fig. 1, curve B). Thus, a good antibody
inhibition study establishes not only the involvement but also the
quantitative importance of a particular cytochrome P450 in the
metabolism of the NME. When it is desirable to obtain information
regarding the variability of cytochrome P450 involvement, particularly
when more than one cytochrome P450 enzymes are involved, similar
studies can be carried out with a panel of human liver microsomal
preparations. Frequently, one can demonstrate a wide range of
involvement of specific cytochrome P450 in the metabolism of a particular
drug with microsomes from different donors [23].
Although specific chemical inhibitors for individual human cytochrome
P450 are rare, isoform-selective inhibitors are generally available at most
pharmaceutical laboratories and are valuable when properly used. Table 1
lists preferred probe substrates and inhibitors for individual cytochrome
P450 enzyme [21]. Similar to antibody inhibition studies, chemical
inhibition studies can be carried out first with a single inhibitor
concentration (known to give strong inhibition with probe substrates) to

94 Lu and Huang
FIGURE 1 Inhibition of human liver microsomal drug metabolism by antibodies

against cytochrome P450. Curve A depicts the strong inhibition of compound A
metabolism by anti-CYP3A4 antibodies. The steep inhibition slope at low antibody
concentrations indicates high potency of this antibody preparation. Maximum
inhibition at higher antibody concentrations indicates that greater than 90% of the
metabolism of compound A is mediated by CYP3A4 in this pooled human liver
microsomal sample. Curve B shows the inhibition of compound A metabolism in
human liver microsome by a different anti-CYP3A4 antibody preparation. The
shallow inhibition slope indicates that either this antibody has a low potency against
CYP3A4 or it cross-reacts with another cytochrome P450. No conclusion can be
made regarding the role of CYP3A4 in the metabolism of compound A. Curve C is
the control experiment showing lack of inhibition of compound A metabolism by
pre-immune IgG. Curve D depicts the inhibition of the metabolism of compound B
by anti-CYP3A4 antibodies. The steep inhibition slope is noted at low
concentrations of this potent antibody. CYP3A4 is responsible for 50% of the
determine which probe inhibitors significantly inhibit the metabolism of the

NME, followed by a more detailed study involving a series of
concentrations of the inhibitors. As shown in Fig. 2 (curves A and B), a good
chemical inhibitor selective for a given cytochrome P450 isoform should
give strong inhibition (a steep inhibition slope) in the metabolism of an
NME at low inhibitor concentrations and reach maximum inhibition at
higher inhibitor concentrations so that the quantitative involvement of this
cytochrome P450 isoform in metabolism can be established. Gradual
increase in inhibition with a wide range of inhibitor concentrations (i.e., a
shallow inhibition slope, Fig. 2, curve C) would suggest that the inhibitor
either has low potency toward the particular cytochrome P450 or it acts as a
poor substrate of the enzyme. In this case inhibition results from the study
have limited values. When studies are carried out using a panel of human

In-vitro Drug Metabolism Studies
TABLE 1 Recommended in vitro Probe Substrates and Inhibitors for CYPs (Ref. [21])
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96 Lu and Huang
FIGURE 2 Inhibition of human liver microsomal drug metabolism by a chemical

inhibitor of CYP3A4. Curve A depicts the strong inhibition of compound A
metabolism by this inhibitor. The steep inhibition slope at low inhibitor
concentrations indicates that this inhibitor of CYP3A4 is very potent. CYP3A4
contributes to approximately 90% of the metabolism of compound A in this pooled
microsomal preparation. Curve B shows that CYP3A4 contributes to 50% of the
microsomal metabolism of compound B. Curve C depicts the shallow inhibition
slope indicating poor inhibition of the metabolism of compound C even at high
inhibitor concentrations. No conclusions can be made regarding the role of
CYP3A4 in the metabolism of compound C.
liver microsomal preparations, different degrees of maximum inhibition in

metabolism provide information regarding the variability of specific
cytochrome P450 involvement in the metabolism of the NME among
individual subjects.
Recombinant Human Cytochrome P450 Enzymes. Microsomes
containing individually expressed human cytochrome P450s provide a
different approach for cytochrome P450 reaction phenotyping. This
approach establishes the intrinsic capability of the individual cytochrome
P450 in the metabolism of an NME, in the absence of other cytochrome
P450 species. If one or more cytochrome P450 species are involved in an
NME’s metabolism, it is important to examine the contribution of each
cytochrome P450 to human liver microsomal metabolism using inhibitory
antibodies or chemical inhibitors. Sometimes, a recombinant cytochrome
P450 found to be involved in an NME’s metabolism, based on a
recombinant enzyme study, may later be shown to play little or no role in
liver microsomal metabolism of the drug in the presence of other
cytochrome P450s, based on an inhibition study. Furthermore, for these
cytochrome enzymes for which activities are observed initially, a
determination of the enzyme kinetics (Km and Vmax) may be warranted so
that the intrinsic clearance and the relative importance of these different

cytochrome P450 species contributing to the metabolism of the NME can be

evaluated [26–28].
Correlation Analysis. Using this approach, the drug is incubated with a
panel of human liver microsomes (preferably more than 10 preparations)
and the reaction rates of an NME determined in each preparation are
correlated with the reaction rates of a cytochrome P450 probe substrate
measured in the same microsomal preparation. If a particular cytochrome
P450 is responsible for the metabolism of the NME, a high correlation
should be observed between the metabolic rates of the drug and the marker
substrate. However, this type of correlation analysis appears to be less
reliable in identifying specific cytochrome P450 enzymes responsible for the
metabolism of an NME. For example, Weaver et al. [29] reported that
58C80 hydroxylation is catalyzed by CYP2C9 based on inhibition and
recombinant cytochrome P450 studies; however, there is no correlation
between 58C80 hydroxylation and CYP2C9 probe substrate activity
(r=0.023). In another study, Heyn et al. [30] reported that although high
correlations between S-mephenytoin N-demethylation and CYP2B6
(r=0.91), CYP2A6 (r=0.88), and CYP3A4 (r=0.74) were observed, other
approaches showed CYP2B6 to be the major enzyme responsible for S-
mephenytoin N-demethylation while CYP2A6 and CYP3A4 played no
significant role in this reaction.
Cytochrome P450 Inhibition

It is important to examine if an NME is an inhibitor of cytochrome P450s
not involved in the metabolism of the drug. For this type of study, the effect
of NME on the metabolism of probe substrate for each of the individual
cytochrome P450 (see Table 1) is evaluated, usually in human liver
microsomes, although individual recombinant human cytochrome P450
enzymes have also been used. The incubation conditions should be such that
initial rates could be measured. To determine the Ki value for any specific
cytochrome P450, at least four to five probe substrate concentrations and
two to three NME concentrations should be used in the assays. Substrate
concentrations should cover a wide range (preferably 10–20-fold) with the
number of concentrations evenly distributed below and above the Km value.
The importance of proper selection of both substrate and inhibitor
concentrations in these studies is well illustrated in the paper by Madan et
al. [22]. The rates of metabolite formation of probe substrate are
determined in the presence and absence of the NME inhibitor and the data
are displayed in graphical representation to determine Ki and the type of
inhibition [22]. Substrate-dependent inhibition has been reported earlier for
CYP3A [49, 51]. Two or more substrates may be needed when evaluating
inhibitors of CYP3A using in vitro methods [21, 47, 49]. Because of

98 Lu and Huang
significant solvent effects (particularly when concentration >1%) reported

for various CYP enzyme studies, low solvent concentrations should be used
in these in vitro studies [47].
In addition to reversible inhibition, time-dependent inhibition of
cytochrome P450 activity by a drug candidate may also be examined to
determine if the NME is a mechanism-based inhibitor. For this type of study,
an NME, at various concentrations (covering a 10–20-fold range), is
preincubated with human liver microsomes with and without NADPH for
various lengths of time (e.g., 0, 10, 20, 30, 45, and 60min) to allow the
generation of reactive metabolites that inhibit cytochrome P450 activity
irreversibly or quasi-irreversibly [22]. At various incubation time points, an
aliquot of the samples is removed and diluted several folds with fresh assay
buffer. The activity of the remaining cytochrome P450 is determined by the
reaction rates of a probe substrate, and the data are displayed in graphical
representation to determine the Ki and Kinact values [22, 31].
If an NME and clinically co-administered drugs are metabolized by the
same cytochrome P450 isoform, inhibition of this cytochrome P450 can
lead to the accumulation of either of the drugs and thereby cause potential
serious drug-drug interactions. This potential can be evaluated using an in
vitro system of human liver microsomes in the presence of both the drugs.
The importance in the proper use of concentrations of either of the drugs is
as described in the preceding section. The Ki value for either of the drugs can
be determined and the potential of drug-drug interaction of co administered
drugs can be evaluated.
Cytochrome P450 Induction

Cytochrome P450 induction represents another mechanism for
metabolismbased drug-drug interactions, although it is much less common
than inhibition-mediated interaction events. Drug treatment can result in
the induction of cytochrome P450 responsible for its own metabolism (i.e.,
auto-induction) or other cytochrome P450s responsible for the metabolism
of co-administered drugs. The major effect of cytochrome P450 induction is
the alteration of drug efficacy and safety over time due to increased
clearance of therapeutic agents resulting in decreased parent drug
concentrations and increased metabolite levels.
To determine if an NME is a cytochrome P450 inducer, the compound, at
several concentrations, is incubated with primary human hepatocytes for
two to five days, and the metabolic rates for probe substrates of individual
cytochrome P450 (generally CYP1A2, 2C9, 2C19, and 3A) are measured
[32, 33]. The NME concentrations should be relevant to its therapeutic
range or, if the theoretical range is not known, a pilot study covering two to
three orders of magnitude may be appropriate. The enzyme activity is

considered to be the most relevant measure while mRNA and Western blot
analyses are useful primarily for mechanistic interpretation [21, 50]. In view
of the individual variability in cytochrome P450 induction, primary human
hepatocytes prepared from at least three individual donor livers should be
used to obtain reliable results. Appropriate positive controls (e.g.,
omeprazole for CYP1A2 induction, rifampicin for 2C9, 2C19, and 3A4
induction) should be included in the study.
In addition to primary human hepatocytes, other in vitro methods such
as receptor ligand assay and reporter gene assay have also been used to
evaluate the intrinsic induction potential of drug candidates [13, 32, 34]. A
positive result of the in vitro induction study can help design clinical trials to
determine if induction is likely to occur at clinical doses and if the extent of
induction may result in significant drug-drug interactions.
Transferases
If an NME is primarily metabolized by a noncytochrome P450 enzyme, it
may become necessary to identify the specific enzyme form responsible for
the metabolism of the compound, particularly if a co-administered drug is
also biotransformed by a similar metabolic pathway and the same enzyme.
However, for enzymes such as flavin-containing monooxygenases,
monoamine oxidases, epoxide hydrolases, glucuronosyl transferases (UGT),
sulfotransferases, methyltransferases, acetyltransferases, and glutathione-S-
transferases, analytical tools are generally not available for carrying out
reaction phenotyping experiments. For example, specific or highly selective
probe substrates and inhibitors are still not available for most of these
enzymes. In addition, antibodies against many of these enzymes are often
noninhibitory so that antibody inhibition experiments can not be performed
to identify the specific enzyme form(s) involved in the metabolism of an
NME. For some of the enzymes, recombinant isoforms remain the only tool
for reaction phenotyping.
When a drug molecule contains functional groups such as—OH,—
NH2,—SH or—COOH, glucuronidation often represents the most
important pathway for its clearance. Therefore, considerable attention has
been paid to UGT reaction phenotyping and its role in drug-drug
interactions [35, 39]. At the present time, highly selective chemical
inhibitors and inhibitory antibodies for individual UGT isoforms are not
available. The only method available to identify the specific isoform
responsible for the metabolism of a drug is to conduct a study with
recombinant UGT enzymes. In addition, a study using a combination of
drugs in human liver microsomes or recombinant system may be valuable in
order to determine if one drug inhibits the metabolism of the other drug or if
mutual inhibition occurs.

100 Lu and Huang
In the literature, there are limited clinical data on UGT-dependent drug-

drug interaction [35], either because of the generally high Km and Ki for
UGTs (therefore low intrinsic clearance and low interaction potential) or
due to the lack of clinical studies designed to address UGT-dependent drug-
drug interactions. Further studies are needed to evaluate the clinical
significance of UGT-dependent drug-drug interactions.
Transporters
It has become increasingly evident that drug transporters, such as P-
glycoprotein, play an important role in the absorption, distribution, and
excretion of many drugs [36–38, 40]. Many substrates, inhibitors, and
inducers of CYP3A4 are also substrates, inhibitors, and inducers of P-gp
[40–45]. Drug-Drug interactions involving transporters, particularly P-
glycoprotein, have become the new focuses in drug discovery and
development. When drugs compete for the same binding sites on the P-
glycoprotein molecule, drug-drug interactions can occur.
To determine if an NME is a substrate of P-glycoprotein and whether the
compound acts as an inhibitor of P-glycoprotein, various in vitro systems,
such as Caco-2 cells, cDNA-transfected Madine-Darby canine kidney cells
and LLC-PK1 pig kidney cells, and derivative cells containing MDR1 (L-
MDR1) can be used. Many studies use digoxin and vinblastine as in vitro
probes and fexofenadine and digoxin as in vivo probe substrates of P-
glycoprotein. The experiments are usually carried out under linear
condition, and the substrate concentrations are at or below their Km values.
Although ATPase and calcein-AM assays have been used, it appears that the
efflux assay (also known as the bi-directional permeability assay) is the
method of choice for evaluating compounds [38, 41].
At the present time, the in vitro methodologies have not been
standardized for the identification of substrates and inhibitors for P-
glycoprotein and other transporters. Prediction of the in vivo drug-drug
interactions from in vitro studies is still problematic. It is expected that more
selective probe substrates and inhibitors will be available for P-glycoprotein
and other transporters (e.g., OATP, MRP, BCRP) in the future, and that our
ability to predict drug-drug interactions in vivo at the transporters level will
be greatly improved.
REGULATORY CONSIDERATIONS
Evaluation of an NMEs drug-drug interaction potential is an integral part
of the regulatory review prior to its market approval [1, 7]. The clinical
pharmacology and biopharmaceutic review of an NDA focuses on key
questions relevant to the review and integrates information across various

studies [46]. For example, in addition to questions addressing how the

following intrinsic factors (age, gender, race, weight, height, disease,
genetic polymorphism, pregnancy, and organ dysfunction) may influence
exposure and/or response, the reviewers also ask questions related to
extrinsic factors:
• What extrinsic factors (co-administered drugs, herbal products,

diet, smoking, and alcohol use) influence exposure and/or
response and what is the impact of differences, if any, in
exposure on pharmacodynamics of an NME?
• Based upon what is known about exposure-response
relationships and their variability, what dosage regimen
adjustments, if any, do you recommend for each of these factors?
Among drug-drug interaction questions, the following may be addressed via

in vitro studies:
• Is there an in vitro basis to suspect in vivo drug-drug interaction?

• Is the drug a substrate of CYP enzymes?
• Is the drug an inhibitor and/or an inducer of CYP enzymes?
• Is the drug a substrate and/or an inhibitor of P-glycoprotein
transport processes?
• Are there other metabolic/transporter pathways that may be
important?
Depending on the answers to the above questions, additional studies may be

conducted to fully assess the interaction potential of an NME with other
drugs, herbal products, and/or food/juices. Figure 3 illustrates one
algorithm in the evaluation of CYP enzyme-based drug-drug interactions of
an NME; starting with in vitro evaluations of the metabolic profile and the
CYP enzyme-modulating effects of the NME using human enzymes. Based
on the outcomes of these in vitro evaluations, which are reviewed along with
additional in vivo clearance information, further clinical studies may be
conducted (Fig. 3).
The appropriate use of in vitro metabolism and drug interaction
information can provide the basis for the design of subsequent in vivo
studies, or obviate the need for further in vivo studies, as illustrated in the
following two cases. For example, Drug A’s effects on various cytochrome
P450 enzyme activities have been evaluated with the following probe
reactions (phenacetin O-deethylation for CYP1A2; tolbutamide 4'-
hydroxylation for CYP2C9, S-mephenytoin 4’-hydroxylation for
CYP2C19, bufuralol 1'-hydroxylation for CYP2D6 and testosterone 6ß-
hydroxylation for CYP3A) using human liver microsomes. The data show

102 Lu and Huang
FIGURE 3 An algorithm for evaluating drug-drug interactions [21].
that Drug A does not inhibit CYP1A2, CYP2C9, CYP2C19, and CYP2D6
at concentrations 100-fold the mean steady state Cmax level achievable
after the administration of the highest proposed clinical dose. Based on this
information, no further in vivo studies on Drug A’s inhibitory effects on
CYP1A2, 2D6, 2C9, and 2C19 will be needed. Drug A inhibits CYP3A.
Further analysis indicates the Ki value to be 1/100 of the Cmax level;
suggesting Drug A to be a strong CYP3A inhibitor. A follow-up clinical
study with oral midazolam administration confirmed its effect on substrates
of CYP3A. The focus of the clinical evaluation on CYP3A has provided data
useful for risk/benefit evaluation of Drug A and subsequent product
labeling. Similarly, Drug B has been evaluated using in vitro methods and
shown to have Ki values in the following rank order:
CYP1A2=CYP2C9>CYP3A>CYP2C19>CYP2D6. As many of these I/Ki
ratios fall within the gray area between “low risk” and “high risk” (21), an
in vivo study focused on CYP2D6 was performed. By focusing on the CYP
enzyme that appeared to be affected most by Drug B, the lack of interaction
from this latter in vivo study would eliminate the need to study Drug B’s
effects on the other CYP enzymes.

LABELING
In a proposed revision of physician labeling format and content, significant

(or evidence of no) drug-drug interactions would appear in the Highlights
section, in addition to having this information in the main body of the labeling
[48]. In vitro and in vivo information on the metabolic pathways and
metabolites, including contribution of specific enzymes, and known or expected
effects of inducers or inhibitors of the pathway, is described in the clinical
pharmacology section of the labeling. Any information on pathways or
interactions that have been ruled out by in vitro data is also included in this
section. Important clinical consequences of this information would be placed
in drug interactions, warnings, precautions, boxed warning, contraindications,
and dosage and administration sections of the main labeling, as appropriate.
Examples of appropriate labeling language are provided in italic below:
[Case 1] In vitro interaction has been studied for the new drug
and no interactions have been demonstrated; no in vivo studies
have been conducted to confirm or refute the in vitro finding.
In vitro drug interaction studies reveal no inhibition of the
metabolism of the new drug by the CYP3A4 inhibitor
ketoconazole. No clinical studies have been performed to evaluate
this finding. However, based on the in vitro findings, a metabolic
interaction with ketoconazole, itraconazole, and other CYP3A4
inhibitors is not anticipated.
Recent examples, such as rosiglitazone (inhibitory effect on CYP enzymes),
and sildenafil (inhibitory effects on CYP1A2, 2C9, 2C19, 2D6, 2E1, and
3A4), are listed in Table 2.
[Case 2] Through in vitro investigations, specific enzymes have
been identified as metabolizing the test drug, but no in vivo or in
vitro drug interaction studies have been conducted.
In vitro drug metabolism studies reveal that the new drug is a
substrate of the CYP ____ enzyme. No in vitro or clinical drug
interaction studies have been performed. However, based on the
in vitro data, blood concentrations of the new drug are expected
to increase in the presence of inhibitors of the CYP ____ enzyme
such as _____, _____, or.
Recent examples, such as pimozide (substrate of CYP3A, ventricular
arrhythmia observed in patients also taking CYP3A inhibitors, macrolide
antibiotics) and Ketoconazole are listed in Table 2.
Recently approved product labels have reflected the increased
understanding of metabolic pathways and consequences of drug

104
TABLE 2 Labeling Examples of Metabolism and Drug-Drug Interaction Information
Lu and Huang
In-vitro Drug Metabolism Studies
TABLE 2 Continued.
105
106 Lu and Huang
interactions by health care practitioners. Newer labels frequently include

in vitro parameters evaluating the drug’s effect on specific cytochrome
P450 metabolism and the clinical consequences of the changes in these
enzyme activities have on co-administered drugs. In addition, the labels
also include the influence of concomitantly administered drugs on the
drug itself. Table 2 lists some examples of the labeling language based on
in vitro information. Less frequently included in the labels today are
transporter information and metabolic interactions based on other
noncytochrome P450 enzymes. As the science progresses and technologies
in the evaluation become standard, future labeling should include these
other types of information.
SUMMARY
As many of the new drugs are to be indicated for patients who receive other
drugs or biologies, it is necessary to know the drug interaction potential
early on in the development. For compounds eliminated by a single
pathway, there is a high probability of drug interaction. The appropriate use
of in vitro metabolism (including isozyme characterization) and drug
interaction information can provide the basis for the design of confirmatory
in vivo studies or obviate the need for further in vivo studies. Further
improvement in the in vitro methodologies evaluating other,
noncytochrome P450-based metabolilsm/drug interactions and
transporterbased interactions should improve our abilities to assess drug-
drug interactions for risk/benefit evaluation during drug development and
regulatory review.
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40. Cvetkovic, M.; Leake, B.; Fromm, M.F.; Wilkinson, G.R.; Kim, R.B. OATP and
P-glycoprotein Transporters Mediate the Cellular Uptake and Excretion of
Fexofenadine. Drug Metab. Dispos. 1999, 27 (8), 866–871.
41. Hochman, J.H.; Yamazaki, M.; Ohe, T.; Lin, J.H.; Evaluation of Drug
Interactions with P-glycoprotein in Drug Discovery: In vitro Assessment of the
Potential for Drug-Drug Interactions with P-glycoprotein. Current Drug
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42. Kim, R.B. Drugs as P-glycoprotein Substrates, Inhibitors, and Inducers. Drug
Metabolism Reviews 2002, 34, 47–54.
43. Kim, R.B., et al. Interrelationship between Substrates and Inhibitors of Human
CYP3A4 and P-glycoprotein. Pharm Res 1999, 16 (3), 3944–3948.
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between Intestinal P-Glycoprotein and CYP3A4. J. Pharmacol Exp. Ther. 2002,
300 (3), 1036–1045.
45. Yasuda, K.; Lan, L.B.; Sanglard, D.; Furuya, K.; Schuetz, J.D.; Schuetz, E. G.
Interaction of Cytochrome P450 3A Inhibitors with P-Glycoprotein. J.
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6
Drug Transporters
Xiaoxiong Wei
Jashvant D.Unadkat
University of Washington
Seattle, Washington, U.S.A.
OVERVIEW
Drug transporters have been a rapidly emerging area in biomedical research

for the last 10 years. These drug transporters are proteins located in the
intracellular and plasma membranes making up to 2–3% of body total
proteins. Drug resistance, low bioavailability, high intersubject variability
and gender difference in drug disposition have been linked to drug
transporters [1–3]. When a drug is introduced into the body, the transport
of a drug from the administered site such as the intestine (absorption) to the
target organs such as brain (distribution) and to the organ for metabolism
and excretion in the liver and kidney (disposition and elimination) is an
important process, in which drug transporters play a critical role. Since this
a very broad field, this chapter will discuss the transporters important in
ADME (absorption, distribution, metabolism and excretion) of drugs.
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112 Wei and Unadkat
Terminology
Diffusion is a process utilized by lipophilic drugs that can readily permeate
the cell membrane down a concentration gradient. Diffusion of polar
substances (e.g. nutrients, ions) across the lipid bilayer membrane of cell is
limited because the cell membrane acts as a diffusion barrier to the
movement of substances into and out of the cell. Cells need to be supplied
with polar or charged nutrients (e.g. amino acids, glucose) or to efflux polar
molecules for physiological function (e.g. bile acids excretion into the gut).
Uptake is a process where the solute is translocated by receptor-mediated or
non-receptor-mediated endocytic process (e.g. LDL and transfertin
receptors). Transport is a process where the solute is translocated via a
membrane protein, which requires a conformational change during the
process of translocation. The solute binding site is accessible to only one side
of the membrane at any one time. It can be either facilitated (passive) or
active. The direction of transport can be influx into or efflux from cells.
Channels are tiny pores, which allow ions such as sodium, potassium,
chloride, calcium to pass through the membrane. There can be several
subtypes of an ion channel for a specific ion. For example, there are several
subtypes of potassium channels in cardiac muscle cells. They may undergo
conformational change to open or close to traffic and may have specific
binding sites for selected solutes. They have binding sites accessible from
either side of the membrane. Transport through channels is always
facilitated (equilibrative) and much faster than that mediated by
transporters.
Classification
Classification of drug transporters is mainly based on energy requirement.
Facilitative transporters move solutes of a single class (uniporters) down a
concentration gradient or an electrical gradient (charged molecules only),
which are not energy-dependent, but protein-mediated (e.g., Na +-
independent equilibrative nucleoside transporters). These transporters are
saturable, and mediate the influx and efflux of drugs, depending on the
direction of the concentration gradient. Active transporters can move
solutes against a concentration gradient, which is energy-dependent and
protein-mediated. There are three types of active transporters: primary,
secondary, and tertiary transporters. Primary transporters generate energy
themselves (e.g., ATP binding cassette or ABC of P-glycoproteins).
Secondary transporters utilize energy (voltage and ion gradients) generated
by a primary active transporter (e.g., Na +/K +-ATPase). Secondary
transporters include symporters and antiporters. Symporters translocate
two or more different solutes in the same direction (e.g., Na+-nucleoside

Drug Transporters 113
transporters). Antiporters couple the transport of solutes in opposite

direction (e.g., H +/organic cation exchanger in the kidney). Tertiary
transporters utilize energy indirectly generated by a secondary transporter.
An example is the transport of organic anions into kidney epithelial cells in
exchange for dicarboxylate ions.
Based on ATP dependence, drug transporters can be divided into two
major classes: ATP-binding cassette (ABC) transporters and non-ATP-
mediated transporters.
MAJOR TRANSPORTERS
Since there are many transporters in biological membranes, we will only

discuss those that are important in pharmacokinetics and
pharmacodynamics of drugs.
ATP-Binding Cassette (ABC) Transporters

The nomenclature of ABC transporters was first introduced in 1992 and
refers to superfamily of transmembrane proteins [4]. These membrane
transporters use ATP hydrolysis as energy to transport a large variety of
substrates across cell plasma membranes. ABC transporters are classified
based on the sequence and organization of their ATP-binding domains
(nucleotide-binding folds, NBFs) rather than their functions. The NBFs
contain characteristic motifs (Walker A and B), separated by approximately
90–120 amino acids, found in all ABC transporters. ABC transporters
typically contain two NBFs and two transmembrane domains (TMD). The
TMDs contain 6–12 membrane-spanning α-helices. The prototypical
structure as found in P-glycoprotein (P-gp) consists of 12
membranespanning α-helices and two NBFs. Both ATP binding sites (NBFs)
are essential for proper functioning of P-gp [5].
ABC transporter superfamily is divided into seven subfamilies: ABCA/
ABC1, ABCB/MDR/TAP, ABCC/MRP, ABCD/ALD, ABCE/OABP, ABCF/
GCN20, and ABCG/White. The members of ABC transporters are still
growing. Thus far, a total of 51 members have been identified [6]. The major
ABC transporters are summarized in Table 1. ABC transporters are located
in normal tissues as well as in cancer cell membranes. The genes from three
subfamilies are highly expressed in most tumor cells and are attributed to
drug resistance, including ABCB1/ MDR1, ABCC subfamily genes (MRP1,
MRP2, MRP4, MRP5, MRP6, MRP7), and ABCG2/BCRP gene.
Particularly, three ABC transporter proteins, MDR1, MRP1, and BCRP, are
found overexpressed in almost all cancer cells responsible for resistance to a
large amount of anticancer drugs [7].

114
TABLE 1 Representatives of main ATP-Binding Cassette (ABC) transporters
Wei and Unadkat

Drug Transporters
Note. MDR: multidrug resistance; Pgp: P-glycoprotein; MRP: multidrug resistance-associated protein; BCRP: Breast cancer
resistance proteins.
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116 Wei and Unadkat
P-glycoproteins (P-gp)
Two genes in ABCB subfamily, MDR1 (ABCB1) and MDR3 (also called
MDR2, AECB4) encode P-glycoproteins (P-gp) [8, 9]. Both the protein
products are efflux transporters. However, MDR3 translocates endogenous
phosphatidylcholine as the main function [10, 11]. Generally, P-gp only
refers to MDR1 gene products. P-gp contains 1280 amino acids, which are
translated from 28 exons of their genes [12]. MDR1 and MDR3 are 76%
identical in gene sequence [13]. Two mouse genes Mdrla and Mdr1b
correspond to the human MDR1 gene. The human MDR1 and these mouse
Mdr genes share 88% identity in gene sequence and have similar function.
MDR1 gene was the first cloned in ABC transporter family [14]. P-gp
(MDR1 gene product) is the best-characterized ABC drug efflux pump. P-gp
plays an important role in multidrug resistance to anticancer drugs in cancer
cells and in the transport of hydrophobic substrates including endogenous
compounds such as lipids, steroids, and a wide variety of drugs. P-gp has
been recognized as one of the important systems to affect bioavailability and
disposition of drugs. More details of the function of P-gp will be described
later. MDR3 is mainly expressed in the bile canalicular membrane of the
hepatocytes to transport endogenous phospholipids from the hepatocyte to
the bile. Recently MDR3 was found to transport some hydrophobic drugs
as well [15].
Multidrug Resistance Associated Proteins (MRPs)

These transporters belong to ABCC subfamily and play a significant role in
drug resistance in cancer cells [16]. MRP1 is expressed in tumor cells and
confers resistance to anticancer drugs, such as doxorubicin, daunorubicin,
vincristine, and colchicines [17]. MRP2 is expressed in canalicular cells in
the liver [18]. It functions as the major efflux pump of organic anions from
the hepatocyte into the bile. Dubin-Johnson syndrome is attributed to a
mutation of MRP2 gene [19]. MRP3 protein is expressed primarily in the
liver. Similar to MRP2, MRP3 confers the ability to efflux organic ions [20].
MRP4 gene is expressed at low levels in many tissues [21]. Overexpression
and amplification of the MRP4 gene is found in cancer cell lines resistant to
nucleoside analogues such as azidothymidine monophosphate. Thus, MRP4
may be an important factor in the resistance to nucleoside analogues [22].
Because these drugs are important antiviral and anticancer agents, this has
importance in therapies for HIV1 infection and cancer chemotherapy.
MRP5 gene is ubiquitously expressed in many tissues. It is closely related to
the MRP4 gene and confers resistance to nucleoside analogues [23]. MRP6
gene is principally expressed in the liver and kidney [24]. Human MRP6
protein is present in isolated membranes and can transport glutathione
conjugates including LTC4 [25]. Genetic polymorphism in MRP6 gene has

been linked with abolished transport activity and disease status such as
abnormal lipid levels [26].
Breast Cancer Resistance Protein (BCRP)

BCRP encoded by ABCG2 gene is a half transporter expressed in normal
tissue [5]. BCRP functions as an efflux transporter serving as a cellular
defense mechanism. Indeed, BCRP and P-gp appear to have considerable
overlap in substrate selectivity. BCRP is highly expressed in the trophoblast
cells of the placenta, which may suggest a potential role in the blood-
placenta barrier [27]. BCRP is also expressed in many resistant cancer cell
lines, which may play a major role in multi-drug resistance in response to
mitoxantrone and anthracycline exposures [28, 29]. Inhibition of these ABC
drug transporters represents a potential strategy for preventing the
development of drug-resistance and increasing anticancer drug
accumulation in tumors.
Non-ATP-Mediated Transporters
Several non-ATP-mediated membrane transporter families have been
identified, which include organic anion transporting polypeptides (rodent:
oatp, human: OATP), organic anion transporters (rodent: oat, human:
OAT), organic cation transporters (OCT), and peptide transporters (rodent:
pept, human: PEPT). These transporter families play important roles in the
disposition and elimination of a variety of endogenous substances, drugs,
and their metabolites from the body. The representative members of these
families are summarized in Table 2.
Organic Anion Transport Polypeptide (OATP)

Currently, at least nine human OATPs have been identified [30, 31]. OATPs
are a group of membrane solute carriers with a wide spectrum of
amphipathic substrates [32]. Although some important members of this
transporter family are selectively expressed in human livers, most human
OATPs are expressed in multiple tissues including the blood-brain barrier
(BBB), choroid plexus, heart, intestine, kidney, and placenta [33–38]. Only
some of the OATPs so far identified have been characterized in detail at the
functional, structural, and genomic levels. Many members of this
transporter family represent polyspecific organic anion carriers for
transport of a wide range of amphipathic organic solutes. Depending on
which side of membrane they are located, OATPs may be responsible for
influx or efflux of a wide variety of amphipathic endogenous substances,
drugs, and their metabolites.

118
TABLE 2 Representatives of the Major Human Non-ABC Transporters
Wei and Unadkat

Drug Transporters
Note: OATP: organic anion-transporting polypeptide; OAT: organic anion transporter; OCT: organic cation transporter.
119
120 Wei and Unadkat
Organic Anion Transporter (OAT)
Human OATs play important roles especially in the elimination of a variety

of endogenous substances, drugs, and their metabolites from the liver and
kidney. So far, five OAT members have been identified [39–43]. Structurally,
OATs are membrane proteins with 12 putative membrane-spanning
domains and function as sodium-independent exchangers or facilitators
[44]. OATs are multispecific organic anion transporters, the substrates of
which include both endogenous (e.g., cyclic nucleotides, prostaglandins,
urate, dicarboxylates) and a wide variety of clinically important anionic
drugs, such as ß-lactam antibiotics, diuretics, NSAIDs, anti-HIV
therapeutics, anti-tumor drugs, and angiotensin-converting enzyme
inhibitors [45–48]. The most commonly used model substrate for OAT
studies is paraaminohippuric acid (PAH). Therefore, the OAT system has
alternatively been called the PAH transport system. All members of the OAT
family are expressed in the kidney, while only some are expressed in the
liver, brain, and placenta [49–51]. The OAT family represents the renal
secretory pathway for organic anions and is also involved in the distribution
of organic anions in the body [52]. OAT-K1, together with MRP2 and
OATP1, may contribute to the efflux of organic anions into luminal side of
renal proximal tubules. OAT-K1 is a Na+-dependent transporter system,
whereas OAT2, OAT3, and OAT4 are Na+-independent transporters, whose
function is to uptake organic anions into cells [53]. OATs may play a role in
drug interactions as well. It has been reported that concurrent use of
methotrexate with acidic drugs, such as NSAIDs, ß-lactam antibiotics,
causes severe suppression of bone marrow, which seems to be related to the
competitive inhibition of the renal OAT system [54].
Organic Cation Transporters (OCT)

Three members of OCT have been reported. OCT1, OCT2, and OCT3
transporters are electrogenic, Na +-independent, and pH-independent
facilitated diffusion systems responsible for the uptake of organic cations
into the cells [55]. In small intestine, liver, and segments of rat kidney
proximal tubules, OCT1 is localized in the basolateral membranes of
polarized epithelial cells [56]. The expression of OCT2 is more
tissuespecific. Human OCT2 is detected mainly in the kidney with some
expressed in brain and small intestines [57–59]. Human OCT2 in brain may
help to reduce the background concentration of basic neurotransmitters and
their metabolites [60].

TISSUE AND CELLULAR LOCALIZATION
The tissue distribution of transporters has been studied using different

techniques. Consistent with their potential role in detoxification processes
and physiological functions, transporters are expressed in various tissues as
demonstrated in human normal tissues as well as in human cancer cell lines.
Certain transporters show a more restricted tissue expression pattern
(MDR3, BSEP, OATP-A, OATP-C, and OATP8) while others can be
detected in almost every tissue that has been investigated (e.g., MDR1,
OATP-B, OATP-D, and OATP-E). This indicates that some transporters
have organ-specific functions while others might be involved in more
housekeeping functions.
Intestines
P-gp is expressed in the luminal membrane of intestinal mucosal epithelium.
Several efflux pumps such as BCRP, MRP2, and MRP4 are also highly
expressed in the intestinal mucosal epithelial cells. However, some of MRPs
are expressed at basolateral membrane of intestinal epithelium, such as
MRP1, MRP3, and MRP5 (Fig. 1). The abundance of P-gp expression varies
in different intestinal sections. The expression of P-gp increases with
distance. (The lowest amount of P-gp is located in stomach, highest in colon,
and medium in jejunum/ileum [61], exactly opposite to the expression of
CYP3A4/5.) CYP3A4/5 expression decreases longitudinally [62].
FIGURE 1 Schematic representation of selected ABC transporters in the intestinal

membrane.

122 Wei and Unadkat
Liver
Liver is an important organ for metabolism of numerous endogenous and
exogenous compounds, a process in which many transporters are involved.
Hepatic uptake of organic anions, cations, and bile salts is supported by
transporters in the basolateral (sinusoidal) membranes of hepatocytes
including OATPs, OATs, and OCTs. ATP-binding cassette transporter
proteins in the canalicular membranes of hepatocytes mediate the hepatic
efflux of drugs, bile salts, and metabolites against a steep concentration
gradient from liver to bile, which includes the MDR1 and MDR3, MRP2,
and BSEP. However, MDR3 is mainly responsible for the transport of
endogenous phospholipids though a recent report indicated that MDR3
may transport some drugs [63]. These transporters play essential roles in
transporting, metabolizing, and excretion of bile salts, xenobiotics, and
environmental toxins (Fig. 2).
Kidney
Multiple organic anion transporters play important roles in the elimination
of a variety of endogenous and exogenous compounds, and their
metabolites from the body. Several families of multispecific organic anion
transporters mediating the renal elimination of organic anions have been
identified. Members of the organic anion transporter (OAT), organic anion
transporting polypeptide (OATP), multidrug resistance protein (MRP),
FIGURE 2 Schematic representation of selected drug transporters in hepatocytes.

sodium–phosphate transporter (NPT), and peptide transporter (PEPT)

families have been identified in the renal proximal tubules. Uptake of
–
organic anions (OA ) across the basolateral membranes of renal epithelial
cells followed by efflux into urine across the apical membrane is mediated
by the Na+-dependent organic transporter, OAT1 and the Na+-independent
organic transporter, perhaps OAT3. The function of MRP6 at the
basolateral membrane is unknown. Efflux across the apical membrane of
organic anions is through low-affinity anion exchange and/or facilitated
diffusion, and a Na +-independent ATP-driven system. The luminal
membrane contains various efflux transporter proteins including OATK1/
K2, OAT4, NPT, MRP2, and MRP4. The luminal membrane also contains
various uptake transporters such as OATP1, PEPT 1/2 (Fig. 3).
Brain
The brain is protected against drugs and toxins by the two drug-
permeability barriers: the BBB and the blood–cerebrospinal fluid (CSF)
barrier (BCSFB). The BBB is primarily formed by the endothelium of the
blood capillaries in the brain. P-gp is expressed in the luminal plasma
membrane of capillary endothelial cells and plays a significant role in
restricting the brain permeability of drugs [64].
FIGURE 3 Schematic representation of selected renal drug transporters.

124 Wei and Unadkat
P-gp is expressed to a great extent in the apical (luminal) plasma

membranes of these capillary endothelial cells, conferring an apical-to-basal
transepithelial permeation barrier to drugs. MRP1 localizes basolaterally,
conferring an opposing basal-to-apical drug-permeation barrier. Together,
these transporter proteins may coordinate secretion and reabsorption of
endogenous substrates and therapeutic drugs into and out of the central
nervous system [65].
Recently, some other transporter proteins including MRPs, OATP, and
OAT have been also reported to exist in the BBB and the BCFSB [66, 67].
Placenta
P-gp is expressed at the brush border membrane of the syncytiotrophoblast.
The expression appears to be higher early in gestation compared with term
placenta [68, 69]. Absence or pharmacological inhibition of placental P-gp
profoundly increases fetal drug exposure. Intravenous administration of
radioactive digoxin, saquinavir, and paclitaxel to pregnant dams resulted in
2.4-, 7-, or 16-fold more drug in fetuses with mdrla (-/-)(-/-) 1b (-/-)(-/-) than
the wild-type fetuses. Placental P-gp could be completely inhibited by
PSC833 or GG918 when given to heterozygous dams indicating that the
placental drug-transporting P-gp is of great importance in limiting the fetal
penetration of various potentially harmful or therapeutic compounds, and
demonstrate that this P-gp function can be abolished by pharmacological
means [70].
The mRNA levels of various transporters in rat placenta were assessed
during late-stage pregnancy. Sixteen mRNAs of various transporters were
expressed in placenta at concentrations similar to or higher than that in
maternal liver and kidney. They include Mdrla and 1b, Mrpl, Mrp5, Oct3
and Octn1, Oatp3, and oatp 12 [71]. The abundance of these mRNA
transcripts in placenta suggests a role for these transporters in placental
transport of endogenous and exogenous compounds. In human placenta,
OATP-B has been detected in the trophoblast at the basal membranes where
it may play a role in transporting natural substrates (e.g., steroid hormone
conjugates) from the fetal circulation into the trophoblast [72].
FUNCTION OF P-GLYCOPROTEINS
P-glycoprotein is the product of multidrug resistance gene family, MDR1

and MDR3. P-gp encoded by MDR3 is expressed at the canalicular
membrane of hepatocytes and is responsible for transporting phospholipids
into bile ductules although a recent report has indicated that it may also
transport some drugs. P-gp, MDR1 product, is expressed in many normal

tissues including intestines, liver, brain, placenta, and testis though it was
first discovered from cancer cells as a multidrug resistance protein. P-gp acts
as an efflux pump by translocating substrates from the intracellular to the
extracellular compartment.
Substrates, Inhibitors, and Inducers

P-gp has an ability to transport drugs diverse in chemical structure from
different therapeutic classes (Table 1). Another striking feature is an overlap
in substrates between P-gp and CYP3A4/5. These two substrate-sharing
systems may serve as protective physiological barriers to limit harmful
exposure to exogenous compounds.
Pharmacokinetic Implication
The high expression of P-gp in many tissues has made P-gp an additional
physiological barrier to protect the body from the exposure to toxins and
xenobiotics. Numerous studies have shown that P-gp plays an important
role in the fate of absorption, distribution, metabolism, and excretion of
drugs.
P-gp was first detected in certain cancer cells associated with the
phenomenon of multiple drug resistance (MDR). However, it is now known
that P-gp is highly expressed in normal tissues. In fact, P-gp is located in the
apical domain of the enterocyte of the lower gastro-intestinal tract (jejunum,
duodenum, ileum, and colon), thereby limiting the absorption of drug
substrates from the gastro-intestinal tract. In other organs such as the liver
and kidney, expression of this transporter at the apical membrane of
hepatocytes and proximal tubular cells in kidney results in enhanced
excretion of drug substrates into bile and urine respectively. P-gp is an
important component in the BBB, limiting the CNS entry of a variety of
drug substrates. P-gp is also found in other tissues known to have tissue–
blood barriers, such as placenta and testis.
Absorption
Drug absorption is a collective result from passive diffusion across intestinal

membranes down a concentration gradient, intestinal metabolism, and P-gp
efflux from the epithelial cells into the intestinal lumen. The effect of P-gp on
drug absorption has been demonstrated using Mdr knockout mice and
studies with P-gp inhibitors. Many clinically significant drug interactions
are due to the inhibition of P-gp in the intestines.
After intravenous and oral administration of paclitaxel, the AUC was
twofold and sixfold higher in Mdrla (-/-) mice compared to the wild-type

126 Wei and Unadkat
(wt) mice. Oral bioavailability of paclitaxel in Mdrla (-/-) and wt mice was
35% and 11% respectively. Biliary excretion of the drug was not different
between the two groups of mice. After oral administration, 87 and 2% of
the dose were found in the feces as paclitaxel in wt and mdrl a (-/-) mice
suggesting substantial change in the extent of absorption of the drug when
the effect of P-gp is removed [73].
Oral absorption of paclitaxel was increased when wt mice were cotreated
with P-gp inhibitors, cyclosporine, or SDZ PSC 833. The oral AUC of
paclitaxel was dramatically increased from 735 to 8066ng.h/ml when
PSC833 was administered [74]. Concurrent drug therapy of P-gp inducers
may decrease drug absorption. After two weeks of treatment with rifampin,
the AUC of a single oral dose of digoxin was significantly reduced, due to
the induction of intestinal P-gp [75].
Distribution
As indicated earlier, the blood, brain, and the placental barriers are
obstacles for a drug to reach the privileged compartments of the brain and
the fetus.
After intravenous administration of digoxin and cyclosporine to Mdrla
(-/-)(-/-) and wt mice, the ratio, (-/-):(+/+), of brain concentrations of
digoxin and cyclosporine in these mice was about 35 and 17, while the
plasma concentration ratio was only 1.9 and 1.4 respectively. Thus, mice
without P-gp have increased concentrations of digoxin and cyclosporine in
the brain [76].
Modulation of P-gp may result in an increase in the CSF levels of the
protease inhibitors and this may have clinical implications. The disposition
of protease inhibitors, indinavir, nelfinavir, and saquinavir was studied in
Mdrla (-/-) and wt mice. Labeled compounds were administered
intravenously and orally. After IV administration, there was no significant
difference in plasma concentrations of total radioactivity at 4h, but the
brain concentrations were considerably elevated in the Mdrla (-/-) mice. The
brain concentration to plasma concentration ratio was the highest for
nelfinavir and lowest for indinavir and saquinavir. After oral
administration, radioactivity in the plasma was higher at 4 h in Mdrla (-/-)
mice for all the three drugs [77]. The efflux of protease inhibitors from the
brain in wt mice can be inhibited by the P-gp inhibitor, LY335959 [78].
OC144–093, a novel, extremely potent inhibitor of P-gp, does not inhibit
multidrug resistance-associated protein (MRP1). This compound is not
metabolized by cytochrome P4503A4, 2C. The enhancement of BBB
penetration of antiepileptic drugs (AEDs) can be achieved with co-
administration of OC144–093 [79]. The presence of P-gp in the placenta
limits fetal exposure to several compounds, but inhibition of P-gp can

enhance the fetus concentrations of protease inhibitors and consequently

may aid in the protection of the fetus from HIV infection.
Metabolism
Cytochrome P450s are expressed in the luminal membranes of intestines.

These CYP enzymes are mainly CYP3A4/5 [62, 80–83]. The co-expression
of P-gp and CYP3A4/5 and the interplay between P-gp and CYP3A4/5 in
enterocytes result in longer residence time in enterocytes for drugs,
potentially resulting in reduced bioavailability of certain drugs [84]. Since P-
gp and CYP3A4/5 share common inducers, such as rifampicin and St. John’s
wort [85], increased expression of both systems may result in reduced
bioavailability of certain therapeutic agents.
Excretion
As described previously, P-gp is highly expressed in the hepatic bile canalicular

membrane and renal proximal tubule luminal membrane. Inhibition of P-gp
may result in changes in biliary excretion or renal proximal tubule excretion
or both, depending on pharmacokinetic characteristics of the individual drug.
Digoxin is mainly eliminated by the kidney (~60%) and the rest by biliary
secretion. Its renal clearance is greater than the filtration clearance indicating
secretion of the drug by the kidney tubules. Kidney epithelial cell lines
expressing human MDRI transport digoxin from basal to the apical
membrane, and this transport is inhibited by cyclosporine [86]. In another
cell line expressing MDRI, the potency of inhibition by the azoles decreased
from itraconazole > ketoconazole >fluconazole [87]. A concomitant use of
itraconazole increases the serum concentrations of digoxin. In a study with
ten healthy volunteers, either 200 mg itraconazole or placebo was given
orally once a day for five days. On day 3, each volunteer ingested a single
0.5-mg oral dose of digoxin. Digoxin AUC (0–72) was approximately 50%
higher during the itraconazole phase than during the placebo phase. The
renal clearance of digoxin was decreased by about 20% (P<0.01) by
itraconazole. The decreased renal clearance of digoxin during the itraconazole
phase may explain increased concentrations of digoxin during their
concomitant use due to the inhibition of P-gp-mediated digoxin secretion in
the renal tubular cells [88].
The effects of quinine and quinidine on the biliary and renal clearances of
digoxin were investigated in healthy subjects. Digoxin was given alone and
with concomitant administration of quinine or quinidine. Quinine and
quinidine markedly reduced the steady-state biliary clearance of digoxin by
about 35 and 42% respectively, while the steady-state renal clearance of
digoxin was reduced significantly only by quinidine (29%) [89]. In a study

128 Wei and Unadkat
of the effect of verapamil on the steady-state digoxin plasma concentrations,

biliary and renal clearance of digoxin, the steady state concentration of
digoxin was increased by 44%, and biliary clearance of digoxin was decreased
by 43%, but renal clearance was unaffected, which may indicate that similar
to quinine, verapamil only inhibits the transporters of biliary system [90].
Genetic Polymorphism
Although the genetic polymorphism of human MDR1 gene has been
reported since late 1980s [91, 92], the impact of MDR1 genetic
polymorphism on drug pharmacokinetics was highly contraversial.
Hoffmeyer et al. conducted a systemic screening for MDR1 polymorphism
and detected 15 single nucleotide polymorphisms (SNPs). An SNP in exon
26 of the MDR1 gene, C3435T (a silent mutation with no amino acid
change), was correlated with P-gp protein levels and digoxin plasma
concentrations after oral administration of the drug. Individuals
homozygous for the T allele have four fold lower P-gp expression and higher
digoxin plasma concentrations compared with CC individuals [93].
However, a later report showed the subjects with genotype TT had lower
digoxin plasma concentrations in a much larger subject pool, a result
opposite to the previous report [94]. Additional reports showed that there is
no correlation between the genotype C3435T and pharmacokinetic profiles
of P-gp substrates [95, 96]. There may not be a solid correlation between
genotype C3435T and its phenotype because this may be linked with other
functional polymorphism in the gene.
Additional functional variants of MDR1 have been disclosed. The
functional relevance of nonsynonymous SNP (G2677T, Ala893Ser) in exon
21 was reported. In vitro expression of MDR1 encoding Ala893 or a
sitedirected Ser893 mutation indicated the enhanced efflux of digoxin by
cells expressing the MDR1-Ser893 variant. In vivo functional relevance of
this SNP was assessed with the P-gp drug substrate fexofenadine. Subjects
with homozygous Ala893 showed higher fexofenadine plasma exposure than
those with homozygous Ser893 [97].
So far, at least 30 SNPs have been reported in the MDR1 gene. Human in
vivo studies on MDR1 genotype-related pharmacokinetics have been
reported. However, results were not always consistent. More work needs to
be done to establish the correlation between the genotype and the phenotype.
Haplotypes of these SNPs may allow a definition of this correlation.
Significance in Drug Development

Because P-gp functions as an efflux pump in cancer cell membranes which
contributes resistance to many anticancer drugs leading to failure of

chemotherapy. Although a few potent P-gp inhibitors are being developed,

the efficacy has not been very satisfactory [98–100]. A challenge facing
pharmaceutical scientists is to develop tumor-specific P-gp inhibitors to
reverse the function of P-gp and to reach adequate accumulation of
anticancer drugs in cancer cells [101]. The same challenge exists for targeted
drug delivery where P-gp expression is abundant. One of the examples is the
delivery of anti-epileptic drugs to the central nervous system [102]. P-gp in
the BBB is the main obstacle to deliver drugs into the central nervous system.
To develop a tissue-targeted P-gp inhibitor or delivery system would provide
an additional strategy to treat many CNS diseases without increased
exposure to peripheral tissues.
The determination of drug candidates as substrates, inhibitors, or
inducers of cytochrome P450s has been a necessary step to meet the
regulatory authorities’ requirements. Lately, whether or not the drug
candidate is a substrate, an inhibitor, or an inducer of P-gp has received a
great attention to because of potential drug interaction issues. Many drugs
are substrates of cytochrome P450 3A and P-gp, and their disposition is
markedly affected by concurrent treatment with inducing agents, such as
rifampin and St. John’s wort. The inducing effects of both these agents have
been reported to substantially decrease plasma concentrations and efficacy
of substrate drugs including cyclosporine [103,104], protease inhibitors
[105,106], oral contraceptives [107], and digoxin [108]. These drugs are
substrates of cytochrome P4503A4 and/or substrates of P-gp. Both rifampin
and St. John’s wort are potent inducers of both CYPs and MDR1 through a
common mechanism that is bound to the pregnane X receptor (PXR) [85].
The screening of PXR ligands has become a useful tool in drug development
to select molecules with a lesser capacity to induce drug-metabolizing
enzymes and MDR1 [109].
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7
Principles, Issues, and Applications of
Interspecies Scaling
Iftekhar Mahmood
Rockville, Maryland, U.S.A
INTRODUCTION
This chapter describes different techniques and approaches to predict

pharmacokinetic parameters from animals to humans during drug
development. These techniques are useful and if used with proper
understanding, it will be time and cost effective. The chapter illustrates the
advantages and the limitations of allometric scaling.
Allometry is based on the assumption that the relationship between
anatomy and physiologic functions is similar among mammalian species [1,
2]. Over the years, allometry has become a useful tool for correlating
pharmacokinetic parameters with body weight from different animal
species. By establishing such a correlation, one can predict pharmacokinetic
parameters in humans which can be useful during drug development.
Interspecies scaling to predict pharmacokinetic parameters in humans can
be performed by two approaches:
i. physiologically based method (PB-PK),

ii. empirical allometric method.
137
138 Mahmood
Physiological method, however, has found only limited use in drug

discovery and development, because this approach is costly, mathematically
complex, and time consuming.
On the other hand, the allometric approach though empirical, is less
complicated and easy to use than the physiologically based method. The
anatomical, physiological, and biochemical similarities among animals can
be generalized and expressed mathematically by the allometric equation.
The allometric approach has been based on the power function, as the body
weight from several species is plotted against the pharmacokinetic
parameter of interest on a log-log scale. The power function is written as
follows:
Y=aWb (1)
where Y is the parameter of interest, W is the body weight, and a and b are
the coefficient and exponent of the allometric equation, respectively. The log
transformation of Eq. (1) is represented as follows:
Iog Y=log a+b log W (2)
where log a is the y-intercept, and b is the slope.

Besides, using the power function to establish a relationship between a
pharmacokinetic parameter of interest and body weight, the power
equation has also been used to establish relationship between body weight
and physiologic parameters such as liver weight, liver blood flow, kidney
weight, kidney blood flow, and glomerular filtration rate of several species
including humans [3].
Using allometric approach, many pharmacokinetic parameters such as
clearance (CL), volume of distribution (V), elimination half-life (t1/2), and
absolute bioavailability (F) from animals to humans have been predicted
[3]. The following sections will describe several allometric approaches to
predict these parameters from animals to humans.
Clearance
Clearance is the most important pharmacokinetic parameter. The
knowledge of clearance is especially very important during drug discovery
or screening process, since drugs which are eliminated quickly may have a
low absolute bioavailability and may not be suitable for further
investigation. Clearance can also play an important role for the selection
of the first-time dosing in humans [as inverse of clearance indicates the
total exposure, area under the curve (AUC) of a drug]. Therefore,
considering the importance of clearance, over the years, a lot of attention

Principles, Issues, and Applications 139
has been focused in order to improve the performance of allometry to

predict clearance. In a given species, clearance can be estimated by the
following equation:
(3)
where AUC is the area under the plasma concentration vs. time curve
calculated by trapezoidal rule and then extrapolated to infinity [4].
A survey of the literature [3] indicates that simple allometry [Eq. (1)]
alone is not adequate to predict clearance in humans from animal data.
Therefore, many approaches have been suggested to improve the prediction
of clearance in humans from animals. These approaches can be summarized
as follows:
Simple Allometry
This approach is based on Eq. (1) or (2), where the clearance of several
species is plotted against body weight.
Maximum Life-span Potential (MLP)
This approach is based upon the concept of neoteny [5] where the clearance
is predicted on the basis of species weight and maximum life-span potential
(MLP).
CL=a (MLP×Clearance)b/8.18×105 (4)
where 8.18×105 (in hours) is the MLP value in humans.

MLP in years is calculated from the following equation as described by
Sacher [6]:
MLP (years)=185.4 (BW)0.636 (W)-0.225 (5)
where both brain weight and body weight are in kilograms. In Table 1, the
MLP values of several species have been presented.
Although Boxenbaum and Dilea [7] mention that neoteny is a trivial
biologic phenomena with no real relationship to the phase I oxidative
metabolism of drugs, MLP appears to be a useful tool that can be used to
predict clearance in humans under specific conditions.

140 Mahmood
TABLE 1 Mean Body and Brain Weight and the Estimated MLP in Several Species
The body weight and brain weight taken from Ref. [8]. The body weight of animals
has been slightly modified as per Ref. [8].
Two-term Power Equation

This approach as suggested by Boxenbaum and Fertig [8] uses a two-term
power equation based on brain weight and body weight to predict intrinsic
clearance of drugs which are primarily eliminated by phase I oxidative
metabolism.
CL=A (body weight)b (brain weight)c (6)
where A is the coefficient and b and c are the exponents of the allometric
equation. Using Eq. (12), one can also predict unbound intrinsic clearance
of drugs.
Product of Brain Weight and Clearance

Mahmood and Balian [9, 10] suggested the use of the product of brain
weight and clearance in order to improve the predictive performance of
allometric scaling for clearance.
CL=(BW×Clearance)b/1.53 (7)
where both brain weight (BW) and body weight (W) are in kilograms.

Mahmood and Balian [9] examined the above mentioned four

methods to predict the clearance of seven antiepileptic drugs in humans
from data obtained from at least three animal species. From the study,
the authors concluded that all the abovementioned methods can predict
clearance with different degrees of accuracy. However, the random use of
these approaches is of no practical value and it is important to identify
the suitability of a given approach. In a separate study, Mahmood and
Balian [10] evaluated three methods (except the two-term power
equation) to predict the clearance of 40 drugs in humans from data
obtained from at least three animal species. In this study, the exponents
of clearance ranged from 0.35 to 1.39. From this study the authors
concluded that there are specific conditions under which only one of the
three methods can be used for reasonably accurate prediction (arbitrarily
selected, if the difference between predicted and observed values is 30%
or less) of clearance:
i. if the exponent of the simple allometry is within 0.55 to 0.70,

simple allometry will predict clearance more accurately than
CL×MLP or CL×Brain Weight.
ii. if the exponent of the simple allometry lies between 0.71 and 1.0,
the CL×MLP approach will predict clearance better compared to
simple allometry or CL×Brain Weight.
iii. if the exponent of the simple allometry is ⭓1.0, the product of
CL×Brain Weight is suitable approach to predict clearance in
humans compared to the other two methods.
It was also mentioned by Mahmood and Balian that if the exponents of the
simple allometry are greater than 1.3, it is possible that the prediction of
clearance from animals to man may not be accurate even using the approach
of CL×Brain Weight, and if the exponents of simple allometry is below 0.55,
the predicted clearance may be substantially lower than the observed
clearance. However, this “rule of exponents” is not rigid and there may be
some exceptions where this rule may not work. Furthermore, one should
also use the scientific judgement when the exponents of simple allometry are
on the borderline (e.g., 0.70 vs. 0.71).
The exponents of allometry are of vital importance and three important
properties regarding the allometric exponents for clearance should be noted:
1. The exponent of clearance will vary with the species used in the
scaling:
For a given drug the exponents of clearance is not universal. The
exponents of simple allometry will depend on the species used in the
allometric scaling. For example, when clearance of ethosuximide was
scaled from mice, rat, and dog [11], the clearance was predicted

142 Mahmood
accurately by a simple allometric equation (exponent=0.51, r=0.880).

The predicted clearance of ethosuximide was 10 mL/min, whereas the
observed clearance was 12mL/min. Using the clearance data from rat,
rabbit, and dog [12], the exponent of simple allometry was 1.01 (r-
0.953). The predicted clearance using simple allometry, MLP, and the
product of brain weight and clearance was 44 mL/min, 15 mL/min, and
10 mL/min, respectively. Scaling of theophylline [10] provided similar
observation as that of ethosuximide. When clearance data were scaled
from mice, rat, rabbit, and dog, the clearance was predicted accurately
by a simple allometric equation (exponent=0.657, r=0.954). Using the
clearance data from rat, rabbit, and dog, accurate prediction of
clearance was only possible by using MLP (exponent from simple
allometric equation was 0.905, r=0.984). This indicates that the
exponents of clearance based on allometric principles depend on the
species used in the scaling and this phenomena will be true for any given
drug. These examples also indicate the importance of the “rule of
exponents.” It is also obvious that the random use of simple allometry,
MLP, or brain weight approach will not help to improve the prediction
of clearance from animals to humans.
2. The exponents of simple allometry have no physiological meaning:
The normalization of clearance by MLP or brain weight is a
mathematical manipulation which may not be associated with the
physiology of the species used in the scaling. As the exponents of the simple
allometry get larger the predicted clearance becomes comparatively higher
than the observed clearance. The predicted clearance values will be on the
order of simple allometry>MLP×CL>brain weight×CL. Furthermore, the
application of MLP and the product of brain weight and clearance is not
limited to the extensively metabolized drugs rather can also be applied to
drugs which are eliminated by renal route.
3. Concept of a fixed exponent of 0.75 for clearance:
The concept of using a fixed exponent of 0.75 for the prediction of
clearance does not seem to be appropriate. From the data published by
Mahmood and Balian [10], it can be seen that the exponents of allometry
range from 0.35 to 1.39. The mean of the exponents is 0.78, which is close
to 0.75, but given the wide range of exponents, it is obvious that using a
fixed exponent of 0.75 will produce serious errors in the prediction of
clearance for many drugs. However, it should be noted that the use of
fixed exponent may be helpful when pharmacokinetic data from only one
species are available. This approach may provide a rough estimate of
clearance but the probability of a large error in the prediction of clearance
is fairly high.

Incorporation of in vitro Data in in vivo Clearance

Human liver microsomes contain different cytochrome P450 (CYP450)
isozymes which are responsible for the biotransformation of xenobiotics
and endogenous substances. With the understanding of the role of
cytochrome P450 in the biotransformation of drugs, it is possible to
characterize the metabolic pattern of a drug. Analysis of the literature
indicates that there are several isozymes (CYP3A4, CYP2D6, CYP2C9,
CYP1A2, CYP2C19) which are responsible for drug metabolism [13].
There are, however, three major isozymes (CYP3A4, CYP2D6, CYP2C9)
which are responsible for the metabolism of almost 90% of drugs [13].
Characterization of drug metabolism in in vitro and extrapolation to in
vivo is gaining momentum. In order to improve the prediction of clearance
in humans, incorporation of in vitro clearance in in vivo clearance has
been proposed. Houston [14] has published a comprehensive review
article on this topic. Lave et al. [15] examined several methods (simple
allometry, product of clearance and brain weight, and in vitro-in vivo
method) to predict clearance of 10 drugs that are mainly eliminated
through hepatic metabolism. In their approach, the authors determined
the rates of metabolism of these drugs in various animal species and
human liver microsomes and hepatocytes. Using the in vitro metabolism
data and combining it with the in vivo data from animals, they predicted
the in vivo clearance in humans using allometric scaling techniques. The in
vivo clearance of each species was normalized by in vitro clearance as
follows:
(8)
Lave et al. [15] concluded that integrating the in vitro data from the
allometric approach with data obtained from at least three animal species
improved the predictions of human clearance as compared to the approach
of simple allometry.
Mahmood [16] reanalyzed Lave’s data [15] and concluded that the
normalization of clearance by MLP (as required based on the exponents)
could have produced the same results as observed when in vitro clearance
was incorporated in in vivo clearance. In the reanalysis of Lave’s data, the
approach of product of brain weight and clearance could not be applied as
the exponents of the simple allometry were less than 1.
In a separate study, Obach et al. [17] used 12 different methods for the
prediction of clearance and concluded that in vitro approach was the best
method for the prediction of clearance. On average the predicted clearance
was within 70–80% of actual values. The authors, however, compared the

144 Mahmood
predicted clearance of the studied drugs using simple allomtery or MLP

randomly.
Indeed, the in vitro approach is one of many attempts to improve the
predictive performance of allometry for the prediction of clearance.
However, the method has not been thoroughly tested and there are very
few published data. Furthermore, the limitations of in vitro approach
should be kept in mind. A definitive disadvantage of in vitro approach is
the necessity of measuring in vitro clearance. The approach cannot be
applied to those drugs which are renally excreted. Therefore, at this time
caution and sound scientific judgement should be used to assess the
reliability of the predicted clearance by in vitro approach. Extensive work
will be needed in this direction before establishing the advantage and
accuracy of in vitro approach in predicting clearance of drugs over other
existing methods.
Number and Suitability of Species for the Prediction of CL

Since testing several species will add time and cost of drug development, it
is always desirable to know the minimum number of species which can
provide a reasonable accurate estimation of pharmacokinetic parameters
in humans for a given drug. Mahmood and Balian [18] investigated
whether clearance in humans can be predicted using two species as
accurately as that of the predictions obtained by using three or more
species (excluding human). Based on the evaluation of 12 compounds the
authors concluded that three or more species are needed for a reliable
prediction of clearance.
Campbell [19] investigated the suitability of a particular species for the
prediction of clearance in humans. He reported that the prediction of
clearance in humans was best predicted when data from rhesus or
cynomolgus monkey were used with MLP. The rat was the next best species
for the prediction of human clearance whereas dog appeared to be a poor
predictor of clearance in humans. Based on limited data analysis, the author
noted that pig also may be a poor predictor of clearance in humans,
especially when MLP is incorporated in the scaling.
Role of Protein Binding for the Prediction of Clearance

Drug-protein binding is a reversible process and drugs may bind to albumin
(weak acidic drugs) and alpha-acid glycoprotein (weak basic drugs). Drug-
protein binding is influenced by a number of factors such as
physicochemical propoerties of drug, concentration of drug as well as
concentration of protein present in the body, the affinity between drug and
protein and disease states such as hepatic or renal impairment.

The kinetics of drug-protein binding can be described by the law of mass

action by the followng equation:
Protein (P)+Drug (D)→Drug-Protein Complex (PD)
An association binding constant (Ka) between drug molecule and protein

can be given as follows:
(9)
The extent of drug-protein complex formed is dependent on Ka. Drugs

strongly bound to proteins have a large Ka values. The number of binding
sites (n) and the association constant (Ka) can be determined by the
following equation:
(10)
where n is the number of the binding sites per protein molecule and r is the
moles of drug bound per mole of protein.
A double reciprocal plot of 1/r vs. free drug concentration (1/D) yields a
straight line whose intercept is 1/n and the slope is 1/nka.
Another graphical technique known as scatchard plot can also provide
binding constants and binding sites. A plot of r/D vs. r yields a straight line
whose intercept is nka and slope is -Ka.
Plasma protein binding vary considerably among animal species which in
turn can influence the distribution and elimination of drugs. Due to this
variability of plasma protein binding among species, it appears logical to
predict unbound clearance in humans from animals. The unbound intrinsic
clearance of many drugs such as antipyrine [8], phenytoin [20], clonazepam
[20], caffeine [21], and cyclosporine [22] with or without normalization to
MLP has been reported in the literature. However, a systematic comparative
study (with the exception of two recent studies) to evaluate if indeed
unbound clearance can be predicted with more accuracy than total
clearance is lacking. Despite this lack of comparative study, it is widely
believed that unbound clearance can be predicted with better accuracy than
total clearance.
Obach et al. [17] in a comparative study attempted to predict the
clearance of several drugs with or without taking protein binding into
consideration. Based on average-fold error (1.91 without protein binding
and 1.79 with protein binding), a slightly improved prediction of unbound

146 Mahmood
TABLE 2 Observed and Total Predicted Clearance (mL/min) of Several Drugs with
or without Considering Protein Binding
*Obtained by multiplying the predicted unbound clearance in humans by free

fraction of drug in human plasma. For example, the predicted unbound clearance of
tamsulosin in humans was 10, 218 mL/min and fu was 0.01. Therefore, the
predicted total clearance in humans was 10, 218×0.01=102 mL/min.
clearance was noted, though for all practical purposes this difference may
not be of any significance.
Mahmood [23] using the rule of exponents compared the total and
unbound clearance of a wide variety of drugs to determine whether
unbound clearance of a drug can be predicted more accurately than total
clearance, and if there is any real advantage of predicting unbound
clearance. The results of the study indicated whether a drug is excreted
renally or by extensive metabolism, unbound clearance may or may not be
predicted any better than total clearance. In his analysis, Mahmood noted
that there are drugs whose unbound clearance can be predicted better than
total clearance or vice versa, but at this time it is not possible to determine a
priori for which drug unbound or total clearance can be predicted better.
Overall, Mahmood’s analysis indicated that correction for protein binding
(unbound clearance) may or may not be helpful for the improved prediction
of clearance in humans from animal data (Table 2).
Prediction of Clearance for Renally Secreted Drugs

Besides hepatic metabolism, drugs can also be cleared by renal route. Renal
clearance is the sum of three processes: glomerular filtration, tubular
secretion, and tubular reabsorption. As a general rule of thumb, renal

clearance greater than 130 mL/min indicates that the secretion mechanism is
involved, whereas a renal clearance less than 130 mL/min indicates tubular
reabsorption. No matter what is the renal clearance of a drug it is possible
that filtration, secretion, and reabsorption processes are simultenously in
operation.
Tubular secretion is an active transport process and is independent of
plasma protein binding but dependent on renal blood flow [24]. Drug
secretion also depends on the affinity of the drug for carrier proteins in the
proximal tubule, the rate of transport across the tubular membrane, and the
rate of delivery of the drug to the site of secretion [24]. All these factors can
be described by following equation:
(11)
where RBF is renal blood flow, fb is free fraction of drug in blood, and CLi is
intrinsic secretion clearance.
Interspecies scaling of drugs for the prediction of clearance may become
complicated due to the differences in the mechanism of excretion of drugs in
different species. It is possible that a drug is extensively secreted in animals
but in humans either drug is not secreted or secretion plays a minor role in
the elimination of drug or vice versa. Mahmood [25], using 10 renally
secreted drugs, demonstrated that it is likely that the predicted total and
renal clearances for renally secreted drugs may be lower in humans than the
observed clearances. The exponents of total clearance of 10 studied drugs
ranged from 0.581 to 0.930. In this study, the predicted total clearance of
seven out of ten drugs was lower by 11–65%. Mahmood and Balian’s
proposed rule of exponents did not help to improve the prediction of total
clearance for these drugs. The predicted renal clearance also did not follow
any particular trend, i.e., for some drugs the predicted clearance was higher
than the observed clearance or vice versa. The prediction of renal clearance
was improved by normalizing the renal clearance by a “correction factor”
for animals which exhibited renal secretion. The “correction factor” was
obtained by the following equation:
(12)
The concept of a “correction factor” is based on the fact that renal secretion
of drugs is based on blood flow. Since the size of the kidneys, body weight,
kidney blood flow, and glomerular filtration rate (GFR) vary from species to
species and can be related by allometry, a correction factor as described in

148 Mahmood
Eq. (12) was found to be suitable in order to improve the prediction of renal
clearance.
Though the proposed approach for the prediction of renal clearance for
renally secreted drugs worked fairly well on the tested drugs, due to small
sample size of drugs used in this evaluation (n=10), more work will be
needed in this direction. Since total clearance of renally secreted drugs could
not be predicted with reasonable accuracy, a method which can improve the
prediction of total clearance for such drugs requires investigation.
Selection of a First-time Dose in Humans Based on

Predicted Clearance
Allometric scaling of a drug in development was performed using oral

clearance of mouse, rat, guinea pig, monkey, and dog. Since the exponent
of the simple allometry was 0.92, MLP approach was considered suitable
for the prediction of clearance in humans. The predicted clearance was
1000 mL/min and 382 mL/min, using simple allometry and the MLP
approach, respectively. Based on the prediction of clearance in humans, an
initial dose of 200 mg was suggested. The human study, however, was
initiated with 15 mg dose. Later, with dose escalation, it was found that
the mean clearance of drug was between 350 and 400 mL/min following
250 and 500 mg dose, respectively, which was very close to the predicted
values.
The above example clearly indicates that allometry can be very useful for
the selection of a first-time dose to humans. In this example, the selection of
a 15-times lower dose to iniate the study was not cost and time effective.
Volume of Distribution
There are three kinds of volumes which are frequently used in the
interspecies scaling.
(a) The volume of distribution of the central compartment (Vc) is

used to relate plasma concentration at time zero (C0) of a drug
and the amount of drug (X) in the body [26]
X=VC×C0 (13)
A small Vc (<3 L) indicates that most of the drug is in the plasma,

whereas a large V c (>7 L) indicates that the drug has
concentrated in the extra vascular space.

(b) The volume of distribution at steady state (Vss) can be estimated

from the following equation:
(14)
where MRT is mean residence time=AUMC/AUC (15)
and AUC and AUMC are area under the curve and area under
the momemt curve, respectively.
(c) The volume of distribution by area (Varea) also known as Vb can
be obtained from the following equation:
(16)
where b is elimination rate constant.
Physiological factors such as plasma protein, tissue binding, total body

water and binding to erythrocytes may effect the distribution of drugs in
the body. Therefore, a drug in the body can be accounted for inside
plasma and outside plasma. The following equation can describe the
relationship:
(17)
where Vp is plasma volume, Vt is tissue volume, and fup and fut are the
fraction of unbound drug in plasma and tissue, respectively. Drugs
extensively bound to plasma proteins (fup < < fut) will have small volume of
distribution.
In an attempt to establish relationship between binding to plasma
proteins and volume of distribution of drugs in animals and man, Swada et
al. [27] investigated the relationship between the volume of distribution
(Vss) and plasma protein binding of b-lactams. Swada et al. [28] also
investigated the relationship between the unbound volume of distribution of
tissues (Vt/fut) and fu (fraction unbound) of nine acidic and six basic drugs in
the rat and in humans. The authors concluded that there was little difference
in Vt/fut of basic drugs between animals and man and that volume in man
from animal data was predicted with more accuracy using Vt/fut than using
volume against fu.

150 Mahmood
Obach et al. [17] used four different methods to predict VSS, and based on
their geometric mean, prediction accuracy concluded that unbound VSS can
be predicted better than the total VSS.
Conceptually there should be a good correlation between body weight
and volume among species and indeed this is the case. Generally the
exponents of volume are around 1.0, which indicates that body weight and
volume are directly proportional. However, this may not be the case for all
drugs, and exponent as low as 0.58 (diazepoxide [29]) has been noted.
Overall, volume of distribution can be predicted in humans from animals
with reasonable accuracy. As noted by Mahmood and Balian [18], unlike
clearance, volume can be predicted in humans with fair degree of accuracy
using two species.
Though literature indicates that V c , V SS , or V b are predicted
indiscriminately in humans from animals, it has been shown by Mahmood
[30] that Vc can be predicted with more accuracy than VSS or Vb. In fact VSS
or Vb may not be of any real significance for the first-time dosing in humans
and can be estimated from human data.
Vc can play an important role in establishing the safety or toxicity for the
first-time dosing in humans. Since an administered dose is always known,
the predicted Vc can be used to calculate plasma concentration of a drug at
time zero (C0) following intravenous administration. This initial plasma
concentration may provide an index of safety or toxicity. Furthermore, Vc
can also be used to predict half-life, if clearance is known (t1/2=0.693 Vc/
CL).
Elimination Half-life and Mean Residence Time

It is difficult to establish a relationship between body weight and half-life (t1/2)
since half-life is not directly related to the physiological function of the body
rather it is a hybrid parameter. A poor correlation between t1/2 and body
weight across the species may give a poor prediction of half-life. Like
clearance, the allometric exponents of half-life using body weight widely
varies. In his evaluation of 18 drugs, Mahmood [30] reported that the
exponents of half-life of these drugs varied from 0.066 to 0.547.
Due to the difficulty in estabishing an allometric relationship between
body weight and half-life, some indirect approaches for the estimation of
half-life have been suggested.
Bachmann [12], Mahmood and Balian [9], and Obach et al. [17] used the
following equation to predict the half-lives of many drugs.
(18)

Though this approach has been found to be suitable for the prediction of
half-life for many drugs in humans, it is also necessary that one must predict
both CL and volume in humans with reasonable accuracy.
Another indirect approach to predict half-life was suggested by
Mahmood [30]. In this approach, first mean residence time (MRT) was
predicted and then the predicted MRT was used to predict half-life in
humans using the following equation:
(19)
The results of this study indicated that MRT can be predicted in humans
with fair degree of accuracy from animal data. The exponents of MRT of
the studied drugs varied from -0.260 to 0.385 (Table 3). The indirect
estimation of half-life using MRT was fairly close to the observed values
(Table 3).
TABLE 3 Predicted vs. Observed MRT and Predicted Half-life from MRT in
Humans from Animal Data

152 Mahmood
Though Eqs (18) and (19) are only true for one compartment model,
both these equations may also be used in a multicompartment system for
prediction purposes.
Species-Invariant Time Methods

In chronological time there is an inverse relationship between the size of the
animal and the heart beat and respiratory rates, in other words, as the size of
the animals increases their heart beat and respiratory rates decrease. On the
other hand, on a physiological time scale, regardless of their size all
mammals have the same number of heart beats and breaths in their lifetime.
Therefore, one may define physiological time as the time required to
complete a species-independent physiological event. Thus in smaller animals
the physiological processes are faster and the life span is shorter.
Chronological time, also known as species-invariant time, can be
transformed into physiological time. Dedrick et al. [31] were first to use the
concept of species-invariant time when they used the pharmacokinetic
parameters of methotrexate in five mammalian species following
intravenous administration as an example. The chronological time was
transformed into physiological time using the following equations:
(20)
(21)
where W is the body weight.

By transforming the chronological time to physiological time, Dedrick
and co-workers demonstrated that the plasma concentrations of
methotrexate were superimposable in all species. They termed this
transformation as equivalent time.
Later, Boxenbaum [20] introduced two new units of pharmacokinetic
time, kallynochrons, and apolysichrons. Kallynochrons and apolysichrons
are transformed time units in elementry Dedrick plot and complex Dedrick
plot, respectively.
Kallynochrons (elementry Dedrick plot):
(22)

(23)
where b is the exponent of clearance.
Apolysichrons (complex Dedrick plot):
(24)
(25)
where b and c are the exponents of clearance and volume, respectively.
Dienetichrons
Boxenbaum [20] introduced a new time unit known as dienetichrons by
incorporating the concept of MLP in physiological time. The
transformation of chronological time to dienetichrons can be obtained by
dividing the X-axis or time by MLP. For example, for elementry Dedrick
plot, X-axis or time was normalized as follows:
(26)
Though some investigators [32–34] have used the concept of species-

invariant time in their allometric analysis, a direct comparison of allometric
approaches with species-invariant time has not been systematically
evaluated.
In a study, Mahmood and Yuan [35] compared the empirical allometric
approaches with species-invariant time methods using equivalent time,
kallynochron, apolysichron, and dienetichrons. Clearance, volume of
distribution, and elimination half-life of three drugs (ethosuximide,
cyclosporine, and ciprofloxacin) were compared using allometric approach
and species invariant time methods. Overall, the species invariant time
method did not provide any improvement over conventional allometric
approach. Especially, the equivalent time approach did not predict plasma
concentrations or pharmacokinetic parameters as accurately as elementry or
complex Dedrick plots. This may be due to the fact that equivalent time
approach uses a fixed exponent of 0.25 for elimination half-life. It should be
noted, however, that the exponent of half-life of drugs is not always 0.25
[30]. The exponents of half-life for ethosuximide, cyclosporine, and
ciprofloxacin in this study were 0.47, -0.24, and 0.04, respectively.

154 Mahmood
Normalization of clearance by MLP provided substantial improvement

in the prediction of clearance for cyclosporine and ciprofloxacin (according
to “rule of exponents” as the exponent of simple allometry was greater than
0.7). The incorporation of MLP in the species invariant time method
substantially underpredicted the clearance and overpredicted the half-life by
more than 20-fold. It was noted by the authors that this inaccurate
prediction of clearance and half-life was mainly due to the prolonged
sampling times in humans following the normalization of MLP. This
increased the AUC and prolonged the half-life of cyclosporin and
ciprofloxacin.
The findings of this study were based on the limited number of drugs
(n=3). Overall, the results of this study indicated that both simple allometry
and species invariant time methods would give almost similar results.
Species invariant time method may be helpful in gaining some insight about
plasma concentrations of a drug but the accuracy of this method in
predicting plasma concentrations in man may not be reliable.
Prediction of Pharmacokinetic Parameters Using Pharmacokinetic

Constants
Besides Species invariant time method, pharmacokinetic constants have
been also used by some investigators to predict plasma concentrations in
humans from animals.
The following equation represents a two-compartment model following
intravenous administration.
C=Ae-at+Be-bt (27)
where A and B are the intercepts on Y-axis of plasma concentration vs. time
plot and a and b are the rate constants for the distribution and the
elimination phases, respectively.
Equation [27] can be used to predict plasma concentrations as well as
pharmacokinetic parameters (using predicted concentrations) in humans
from animal data. Swabb and Bonner [36] and Mordenti [37] predicted
plasma concentrations of aztreonam (one compartment model) and
ceftizoxime (two compartment model) in humans from animal data
using pharmacokinetic constants. Though Swabb and Bonner and
Mordenti successfully used pharmacokinetic constants approach for the
prediction of plasma concentrations and pharmacokinetic parameters,
the suitability of this approach for the prediction of pharmacokinetic
parameters in humans from animal data has not been thoroughly
investigated.

Mahmood [38] compared the predicted pharmacokinetic parameters

of six drugs using either pharmacokinetic constants or conventional
allometric approach. No trend in correlation between body weight and
A, B, or a was found. For some drugs a good correlation between body
weight and these parameters was obtained whereas a poor correlation
was observed for other drugs. Though the predicted values of A and B
were occasionally close to the observed values, the predicted a values
were many folds higher or lower than the observed values which had
substantial effect on the predicted plasma concentrations. Overall, the
use of pharmacokinetic constants to predict pharmacokinetic
parameters in humans from animal data did not provide any
improvement over conventional allometric approach. Like species
invariant time method, pharmacokinetic constant approach may
provide some information about plasma concentrations of a drug but
the accuracy of the method for the prediction of plasma concentrations
in man may be questionable.
Absorption and Absolute Bioavailability

Prediction of absolute bioavailability in humans from animals due to the
differences in the anatomical and physiological features of the
gastrointestinal tract, dietry habits, blood flow through the gut and the liver,
and the enzymatic activity of the metabolizing enzymes, is a complex task.
Some animal models may provide a rough estimate of absolute
bioavailability in humans and such rough estimates can also be of significant
importance to identify problems of absorption and intestinal and hepatic
metabolism in man.
Conceptually it is difficult to justify an allometric relationship between
body weight and absolute bioavailability. Mahmood [39] using direct (body
weight vs. absolute bioavailability) and several indirect approaches
attempted to predict absolute bioavailability in humans from animal data.
Five different methods were used to predict absolute bioavailability in
humans:
i. body weight vs. absolute bioavailability (allometric approach)

ii. F=CL(IV)/CL(oral) (28)
iii. F=1-(CL(IV)/Q) (29)
iv. F=1-(CL(oral)/Q) (30)
v. F=Q/(Q+CL(oral)) (31)
where Q is hepatic blood flow (1500 mL/min). Methods II-V are indirect
approaches. Fifteen drugs were used in this analysis. In Table 4 the

156
TABLE 4 Predicted and Observed Absolute Bioavailability of 15 Drugs using Different Methods
NC=Not calculated because there were only nine drugs available for this method.
NA=Not available. Oral clearance was greater than the liver blood flow (1,500mL/min).
*Method III not included in the analysis. Method I=body weight vs. absolute bioavailability; Method II: F-CL(IV)/CL(oral); Method III:
Mahmood
F=1-(CL(IV)/Q); Method V: F=Q/(Q+CL(oral)).
Reproduced with kind permission of the copyright holder, Drug Metabolism and Drug Interactions (Ref. [39]).

correlation coefficient between body weight and absolute bioavailability,

exponents of allometric equation, and the predicted absolute bioavailability
in humans from animals have been shown. Though for some drugs a good
correlation between body weight and absolute bioavailability has been
obtained, there is uncertainty in the prediction of absolute bioavailability in
humans from animals. Overall, the results of the study indicated that all the
five approaches predict absolute bioavailability with different degrees of
accuracy and are unreliable for the accurate prediction of absolute
bioavailability in humans from animals. Despite uncertainty in the
prediction of absolute bioavailability in humans, the approach may provide
a rough estimate of absolute bioavailability.
Sietsema [40] plotted absolute bioavailability in man against those in
rodents, dogs, and monkeys. The correlation coefficient (r2) for absolute
bioavailability between man and rodent, man and dog, and man and
primates was 0.4, 0.3, and 0.2, respectively. This poor correlation indicates
that absolute bioavailability data in animals may be of moderate use for the
prediction of absolute bioavailability in humans.
In recent years, attempts have also been made to correlate fraction of oral
dose between rat and humans [41]. For the prediction of absorption in
humans, methods such as intestinal permeability in rats [42, 43], jejunal
permeability in humans [42, 43], and caco-2 cell permeability [44] have
been proposed.
Prediction of Maximum Tolerated Dose (MTD)

In phase I clinical trials, not only the selection of the first dose to be
administered to the patients is a challenge but also the issue of dose
escalation is a complex task. A conservative low-dose approach will result in
subtherapcutic or ineffective dose. On the other hand an aggressive dose
escalation may result in producing toxicity. Certain class of drugs, for
example anticancer drugs, are so toxic that for ethical reasons they can not
be given to healthy subjects. Therefore, predicting MTD in humans from
animal data may prove to be highly beneficial. For anticancer agents,
generally 1/10 of the LD10 in mice or 1/3 of the toxic dose level (TDL) in the
dog in mg/m2 is used as the starting dose in phase I clinical trials [45].
Goldsmith et al. [46] reported that the use of 1/3 of the TDL would have
produced significant toxicity in the patients for 5 out of 30 drugs. The
authors further concluded that for a safe starting dose in phase I clinical
trials, not only toxicology data from dog and monkey, but also data from
rat, mice, and tumor-bearing mice should be included. Similary Homan [47]
concluded that there was a 5.9% probability of exceeding the human
maximum tolerated dose (MTD) if the starting dose in clinical trials were 1/
3 of the TDL of large animal species (dog or monkeys). Rozencwig et al. [45]

158 Mahmood
concluded that 1/6 LD10 in the mouse and 1/3 toxic dose low in the dog
corresponds to an acceptable dose in humans provided both preclinical and
clinical data are obtained under identical schedule and compared on a mg/
m2 basis. Mice and dogs may provide different informations for a given drug
but combining data from both species can be helpful in determining the
starting dose in humans for phase I clinical trials [45].
Mahmood [48] using 25 anticancer drugs examined whether or not
MTD can be predicted from animals to humans. The predictive
performance of two different approaches of allometry for the prediction of
MTD was compared in humans from animal data. The two approaches to
predict MTD in humans were: (i) the use of a fixed exponent of 0.75 and the
LD10 in mice; and (ii) the use of LD10 (in case of mice) or MTD data from at
least three animal species (interspecies scaling). The results of the study
indicated that MTD can be predicted more accurately using interspecies
scaling than using a fixed exponent of 0.75. Like clearance, it was noted that
incorporation of mean life-span potential (MLP) can also be used to
improve the prediction of MTD for some drugs. One-third of the predicted
MTD from interspecies scaling can be used as a starting dose in humans.
This approach may save time and avoid many unnecessary steps to attain
MTD in humans.
Prediction of Inhalational Anesthetic Potency Minimum Alveolar

Concentration (MAC)
Interspecies scaling is frequently performed to predict pharmacokinetic
parameters from animals to man and a fair amount of research has been
successfully conducted to correlate body weight with the pharmacokinetic
parameter(s) of interest [5, 49, 50]. However, very little information is
available for the prediction of pharmacodynamic parameters from animals
to man. Travis and Bowers [51] applied the principles of allometry to the
minimum alveolar concentration of several inhalational anesthetics. The
authors found that not only there was a poor correlation between body
weight of animals and the MAC but the slope of the allometry was
statistically not different from zero. Lack of correlation between body
weight and MAC and a slope nearly zero made it almost impossible to
predict MAC in humans.
MAC is defined as the minimum concentration of inhalational anesthetic
agent in the alveolus at steady state which will inhibit a muscular response
to stimulus in 50% of patient population and is expressed as volume percent
required at one atmosphere [52]. Thus MAC represents EC 5o on a
conventional quantal dose response curve.
Using a correction factor, Mahmood [53] attempted to predict MAC
from animals to humans. The MAC values of 10 anesthetics were obtained

from the literature. At least three animal species (excluding humans) were
used in the scaling. Interspecies scaling of MAC was performed using the
following two methods:
i. Using traditional allometric approach, the MAC of each drug

was plotted against the body weight of the species on a log-log
scale and from the resultant equation MAC was predicted in
humans;
ii. MAC in each species was multiplied by a “correction factor”
obtained by adjusting the lung weight of the species based on per
kg body weight. The product of correction factor and the MAC
was then plotted against body weight on a log-log scale.
Using the simple allometric approach, the correlation between body weight
of the species and the MAC was found to be poor. The exponents of the
simple allometry varied from -0.026 to 0.105. The mean of the exponents of
all 10 drugs was 0.027 which was statistically not different from zero. The
error of predicted values ranged from 28–134%. The predicted MAC in
humans was overestimated at least by 50% for six drugs.
On the other hand, incorporation of “correction factor” substantially
improved the correlation between body weight and the MAC. The
exponents of the allometry varied from 0.078 to 0.218. The mean of the
exponents of all 10 drugs was 0.127 which was statistically different from
zero. The error of predicted values ranged from 2–92%. The predicted
MAC in humans was overestimated by 50% for only two drugs.
It is difficult to visualize that there will be a correlation between body
weight and MAC, since a change in a pharmacodynamic parameter may not
simply be a function of change in body weight. The concept of a “correction
factor” for anesthetic gases and vapors is based on the fact that these
anesthtics are administered to patients at appropriate inspired
concentrations. Depth of anesthesia is determined by the concentration of
anesthetic agent in the brain. The rate at which an effective brain
concentration can be acheived depends on the rates of transfer of inhaled
anesthesia from the lung to the blood and from blood to the tissues, the
solubility of the anesthetic from the lungs to the arterial blood, its
concentration in the inspired air, pulmonary ventilation rate, pulmonary
blood flow (change in cardiac output), and the partial pressure of the
anesthetic between arterial and mixed venous blood. Considering all the
abovementioned factors in order to achieve an adequate concentration of an
inhaled anesthetic, it appears lung plays a vital role, as the lung is the site of
drug delivery. Therefore, taking into account that the role of lung in
maintaining an adequate concentration of a given anesthetic in the brain is
vital and since the size of the lung and the body weight varies from species to

160 Mahmood
species, normalization of the lung weight based on the body weight was
found to be suitable for the improved prediction of MAC in humans from
animals. Though data for inhaled drugs other than anesthetics were not
evaluated, the findings of this study may be extended to other inhaled drugs.
The concept of a correction factor for the prediction of a parameter of
interest (especially for a pharmacodynamic parameter) for inhaled drugs
other than anesthetics should be examined.
CONCLUSION
The allometric scaling of pharmacokinetic parameters can be useful to

select a safe and tolerable dose for the first-time administration to
humans. Thus scaling can provide a rational basis for the selection of a
first dose in humans. Therefore, in recent years, interspecies scaling of
pharmacokinetic parameters has drawn enormous attention. Over the
years many approaches have been suggested to improve the predictive
performance of allometric scaling. Though not perfect, these approaches
are of considerable importance to understand and refine the concept of
allometric scaling.
There may be anatomical similarities among species but there are
external factors which will affect the allometric scaling. Experimental
design, species, analytical errors, and physico-chemical properties of drugs
such as renal secretion or biliary excretion may have impact on allometric
extrapolation.
Despite the fact that allometry is empirical and occasionally fails to
perform adequately, further investigation should be conducted to find the
underlying reasons for failure.
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8
Analytical Method Validation
Brian P.Booth
W.Craig Simon
Therapeutic Products Directorate
Health Canada, Ottawa, Ontario, Canada
INTRODUCTION
The purpose of this chapter is to describe the elements of analytical method

validation promulgated by the U.S. Food and Drug Administration (FDA)
for drug development, and to explain the reasoning for each component.
This chapter is intended for individuals who are unfamiliar with analytical
method validation, or new to drug development. Readers who are interested
in more detailed experimental or statistical treatises of specific aspects of
method validation are referred to elsewhere.
Analytical method validation is the process used to determine the
capabilities and limitations of an assay. This process is very important
because the data these assays generate are used to make chemical,
pharmacokinetic, and pharmacodynamic conclusions about drugs. The
ability to make these conclusions is of great importance, because they in
turn are used to support claims regarding the safety and efficacy of new
drugs to be used in human patients. This demonstration of safety and
165
166 Booth and Simon
efficacy of a new drug or therapeutic is required by law in the United States,

Europe, Canada, and Japan. Therefore, the failure to ensure the reliability of
an analytical method, the data it generates, and the resulting conclusions
can raise significant questions about the validity of the drug safety and
efficacy claims.
Analytical method validation has been addressed by the U.S. Food and
Drug Administration, the European Medicines Evaluation Agency (EMEA;
the regulatory body of the European Union), and the regulatory agencies of
other countries such as Australia, Canada, and Japan. As a result of the
efforts of the International Committee on Harmonization (ICH), the
differences in the approaches and requirements to analytical method
validation by different countries have been minimized. However, the reader
is cautioned that there may be different requirements in different countries
and appropriate guidance should be sought for submissions elsewhere. The
remainder of this chapter explains the characteristics of method validation
that are promulgated by the U.S. FDA in the Guidance for Industry entitled
“Bioanalytical Method Validation” [1]. The principles described in the
document were established following a workshop cosponsored by the FDA
and the American Association of Pharmaceutical Scientists (AAPS) in 1990
[2]. The workshop was attended by regulatory scientists, pharmaceutical
industry scientists, and academicians involved in pharmaceutical analysis.
The principles that were developed are described in the original draft
Guidance for Industry, “Bioanalytical Method Validation for Human
Studies” which is currently posted on the FDA internet site [3]. However,
these analytical method validation principles were recently updated at a
second workshop convened by the FDA and AAPS in January 2000 [4].
Additional guidance has been included for the newer analytical technologies
of LC/MS/MS and ligand binding assays such as radioimmunoassays (RIAs)
and enzyme-linked immuno-sorbent assays (ELISAs) [4].
Analytical method development and validation are usually completed
prior to the start of preclinical and clinical pharmacology studies
(bioavailability, bioequivalence, individual, or population pharmacokinetic
studies) of new chemical entities intended for submission to the FDA as New
Drug Applications (NDAs). Analytical method validation is also required
for the development and assay of generic drugs, which are the subject of
Abbreviated New Drug Applications (ANDAs), and veterinary drugs.
Analytical method validations are also required for the Chemistry,
Manufacturing and Controls (CMC) section of NDAs and ANDAs that
describe the chemical quality and stability characteristics of the drug.
However, the FDA Office of New Drug Chemistry issued a separate
Guidance for Industry for the Validation of Chromatographic Methods, and
the reader is referred to this document for specifics regarding CMC issues of
NDAs and AND As [5–7].

Analytical Method Validation 167
TYPES OF ANALYTICAL METHODS
Chromatographic methodologies have proved very useful for drug analysis.

From the mid-1970s to the early 1990s, the most widely used analytical
methodologies in drug development were gas-liquid (GC) and high
performance liquid chromatography (HPLC).
Gas-Liquid Chromatography
In GC, samples are vaporized in the injection port, and sample constituents
are then separated as they are moved along the length of the column by the
carrier gas. Separation of the constituents is achieved because each compound
possesses a characteristic rate of dissolution into the stationary phase and
revolatilization into the mobile phase that is dependent upon the
characteristics of the compound, and the stationary phase used in the method
(see Fig. 1) [8]. The extent of separation can be increased or decreased to
some extent by altering the temperature of the oven in which the
chromatographic column is housed. Some advanced GC systems also
incorporates hardware that allows for variable injection port temperatures
to increase analyte separation. However, the main means of increasing the
separation of the analyte from other sample constituents is the choice of the
stationary phase/column used in the method. As each analyte exits the column,
it is detected and quantified by a detector (e.g., mass spectrometer, electron
capture, flame ionization detectors, etc.). Gas chromatography is generally
characterized by great analytical sensitivity, often as low pg/ml, but it is
limited by the need to volatilize the compounds of interest. Compounds with
high boiling points are difficult to vaporize and cannot be quantified by GC
very readily [8]. For this reason, HPLC has been more widely used.
HPLC
In HPLC, the samples are dissolved in a solvent and injected into the system.
The analytes are then separated from other sample constituents by the
differential rates of dissolution into the mobile phase and the stationary
phase. The rate of this process is a characteristic of the analyte, mobile, and
stationary phases used in the system. Increased or decreased separation can
be obtained by altering the composition of the mobile phase solvent (i.e.,
changing the solvent polarity). Analytes are detected upon exiting the
column by several types of detectors (i.e., UV-VIS, fluorescence, electro-
chemical, mass spectrometers, Fourier Transformed Infrared (FTIR)
detectors). The main limitation with HPLC is the ability to dissolve the

168 Booth and Simon
FIGURE 1 Chromatographic Separation. In GC, compounds are acted on by two

forces: the carrier gas (mobile phase) which sweeps the molecules along the
column (but does nothing to separate molecules), and dissolution of the
compounds into the stationary phase. Separation is accomplished by the
differences in the rate of dissolution of the molecules into and out of the stationary
phase. The circles represent molecules with lower vapor pressures, which spend
more time dissolved in the stationary phase. The circles are held up by the
stationary phase, whereas the molecules represented by the squares have a
higher vapor pressure (lower boiling point), and spend more time in the mobile
phase, which sweeps these molecules out of the column faster than the circles.
Therefore, the squares are swept through the column to the detector faster than the
circles. (The squares have a shorter retention time.) In HPLC, these interactions
are similar. The difference is that a solvent is used in the mobile phase, and it
contributes to the forces that separate the molecules.

sample in a solvent. This difficulty, however, is much less of a problem in

HPLC than sample vaporization is in GC. The limit of detectability is
usually lower with GC than HPLC (10 to 100 times), depending on the type
of detector used. Generally, UV-VIS and fluorescence detectors in HPLC
provide less sensitivity than GC detectors, but electrochemical and mass
spectrometric detectors could provide equivalent sensitivity to GC systems.
LC/tandem Mass Spectrometry

Currently, the most widely used analytical technology is LC/MS/MS.
Traditionally, these systems were cumbersome and difficult to use, but
recent advances in technology and automation have made LC/MS/MS
systems the stalwart of current analytical methodologies. LC/MS/MS
depends on HPLC to separate the analyte from other matrix constituents as
described in the preceding section, but the use of tandem mass spectrometry
allows for the detection of very small quantities of drug, in addition to
generating information about the chemical structure of the analyte which
allows for analyte identification.
Ligand-Binding Assays
In addition to LC/MS/MS, greater use is currently made of nonchromatographic
techniques. The two most prevalent techniques, radioimmunoassays (RIAs)
and enzyme-linked immunosorbent assays (ELISAs) are ligandbinding
techniques. These assays are based on specific or relatively specific antibodies
that are developed for the analyte of interest (see Fig. 2).
RIAs
In a RIA, the analyte is incubated in a buffer with the antibody and a known
quantity of radiolabeled analyte. After incubating these reactants for a
period, the samples are centrifuged and the radioactivity in the bound, pellet
fraction is counted (in some cases, the unbound tracer in the supernatant is
counted instead). As the amount of analyte increases, more radioactive
analyte is displaced and the amount of radioactivity in the pellet decreases.
Therefore, low radioactivity corresponds to higher amounts of actual
analyte in the sample (see Fig. 3).
ELISAs
In an ELISA, the antibody is usually bound to a surface, and linked to some
type of enzymatic reporter system (for instance, horseradish peroxidase).
Typically, the enzymatic reporter systems are linked to the surface of 96-well

170 Booth and Simon
FIGURE 2 RIAs and ELISAs. These assays are ligand-based assays. The triangle
represents the analyte of interest. In the RIA, the analyte displaces the binding of a
known quantity of radiolabeled analyte (triangle with 125I). The oddly shaped
molecule with a triangular edge represents a potential interference, namely a
molecule with a similar hapten as the analyte of interest. In the ELISA, once the
analyte binds the antibody (which is bound to a surface), the enzyme linked to the
antibody is activated to signal the interaction.
plates. Samples are added along with the necessary reactants, and gently
mixed. After a defined period of incubation, the reaction in each well is
“stopped” and the amount of analyte is quantified (often using a
spectrophotmetric plate reader). One of the major drawbacks with
ligandbased assays is antibody binding to nonanalyte entities. This type of
binding will produce overestimates of the analyte quantity. It can be difficult
to determine whether this process has occurred because unlike
chromatography, there is no visual output to assess. Therefore, greater care
has to be taken to ensure that no interference occurs in these types of assays.
ANALYTICAL METHOD VALIDATION
After choosing the best analytical method to be used, which includes the
type of analytical principle (e.g., HPLC), hardware, extraction, and
reconstitution procedures (isolation of the analyte from the sample matrix),
the limitations of the complete assay need to be determined. Analytical
method validation essentially consists of three discrete steps: (1) assessing

FIGURE 3 RIA Standard Curve. The X-axis is the log of the concentration range (1
to 100 units), and the Y-axis reports the amount of radioactive tracer that is bound to
the antibody. As increasing amounts of nonlabeled analyte from the sample are
incubated, increasing fractions of the radioactive tracer are displaced. Therefore,
the curve declines with increasing concentrations of unlabeled analyte.
the limits of the analytical assay, (2) determining the effect of sample
handling, and (3) monitoring assay quality during practical use.
Assessing the Limits of the Analytical Assay

Several aspects are assessed, and these are summarized in Fig. 4. Essentially,
the bioanalyst needs to define a box that is bounded by the upper and lower
limits of acceptable error, and the upper and lower limits of quantification.
Once these limits are defined, we will be confident that experimental
determinations of analyte concentrations that are within this box are
reliable. The specific assay characteristics of interest are as follows.
The Standard Curve (Calibration Curve)

The relationship between drug concentration and the response of the
analytical system needs to be determined. This mathematical relationship
will allow us to later determine analyte concentrations of unknown clinical

172 Booth and Simon
FIGURE 4 Standard Curve. The detector responses to a drug are plotted against
six duplicate concentrations of drug ranging from 5 to 500ng/mL (•). The upper level
of acceptable error in the drug concentrations is represented by the triangles, and
the lower level by the open circles. The ULOQ is 500 ng/ml, and the LLOQ is 5 ng/
ml. The solid line through the actual data was linearly regressed, and generated an
equation for a straight line with the form Y=AX+B, where Y is the machine response,
A is the slope of the curve, X is the drug concentration and B is the intercept on the
y-axis. With the values of A and 8, the value Y for unknown samples is determined
by analysis, and the corresponding concentration is then back-calculated.
samples from the response obtained from the analytical method. The
standard curve of the method is specific for each drug in a specific matrix
(e.g., blood, plasma, urine, cerebrospinal fluid, etc.). If the drug will be
measured in plasma during the clinical study, the standard curve should be
constructed by spiking drug into plasma, and then extracting and analyzing
the concentrations. The use of different solvents such as water or methanol
is not recommended because there may be differing solvent characteristics
(such as solubility, protein binding, etc.), and this could complicate the
interpretation of the data. The drug stock solution must be made in a
solvent, but all subsequent dilutions should be in sample matrix.
If samples will be taken from more than one matrix (e.g., plasma and
urine), then standard curves must be constructed for each. The same is also
true if more than one analyte is to be measured (e.g., parent drug and
metabolite). Although parent and metabolite may be simultaneously
quantified from the same sample, a standard curve for each specific analyte

must be constructed. It is also advisable to incorporate the use of an internal

or external standard in sample preparation, although this step is not a
requirement for method validation. Standard curves should be constructed
with a minimum of six drug or analyte concentrations spiked in the
appropriate matrix (see Fig. 4). Once these standards are measured, the data
should be plotted (response vs. analyte concentration), and the simplest
curve which best fits the data should be generated to describe the
relationship. Zero or blank samples should not be included in the
curvefitting procedure because the assay is characterized by a lower limit of
quantification which is higher than “zero” or no drug, and inclusion of this
point might alter the fit of the curve. Curves generated without weighting of
the data are preferred, but weighting the data is permitted. Usually,
weighting is used in cases where the range in drug concentrations spans
several orders of magnitude, and weighting helps account for the
heterocedasticity in the data. The relationship that is derived is then used to
back-calculate drug concentrations from clinical study samples. The slope
of the curve indicates the sensitivity of the assay; small changes in
concentration that induce large changes in response indicate a sensitive
method [9].
Range
The range of the standard curve should cover the expected range of
concentrations that will be covered in the clinical study. The range is
bracketed by the lower limit of quantification (LLOQ or LOQ, see Fig. 4;
data below LLOQ are often reported as BQL—below quantification limit)
and the upper limit of quantification (ULOQ, see Fig. 4). Extrapolation of
drug concentrations beyond either limit is not acceptable. Concentrations
below the LLOQ cannot be measured, unless further analytical
development is conducted. One possible approach is validating the use of
larger sample volumes at concentrations near the LLOQ [9]. Drug
concentrations that are beyond the ULOQ of the assay should be diluted
and reassayed. Determining the effect of sample dilution is helpful. Sample
dilutions should be conducted using like matrix, e.g., plasma for plasma
samples, urine for urine samples, etc. Use of a nonlike matrix can alter the
physicochemical conditions acting on the analyte, causing nonlinearity
which may lead to errors in sample quantification.
LLOQ
The LLOQ is the lowest concentration that can be reliably measured with
the assay. The LLOQ is often confused with the lower limit of detection
(LLOD; LOD). The LLOD is the lowest response that can be detected by

174 Booth and Simon
FIGURE 5 LLOQ. LLOD, is defined as two times the background noise. LLOQ is
defined as five times the background noise.
the analytical hardware. It is usually defined as the signal that is two or

three times higher than the background noise (signal to noise ratio of 2 or
3; see Fig. 5). LLOQ is often defined as some multiple of the LLOD (e.g.,
three or five times higher). However, the LLOQ is defined in the FDA
Guidance as the response that is at a minimum of five times higher than
the response to a blank sample, which is slightly ambiguous because it is
not necessarily related to the minimum ability of the detector to measure a
signal. The EMEA adopted the ICH definition, which defines the LLOQ as
10 times higher than background [10]. In Canada, the LOQ is deemed
acceptable if the precision has been adequately demonstrated for that
concentration.
Selectivity
The selectivity (also referred to as specificity) is the ability of the assay to
measure the drug or analyte without interference from other constituents
in the sample matrix. In chromatographic systems, selectivity is
demonstrated by comparing the detector response in the presence of drug,
to a blank sample of plasma that was not exposed to the analyte (see Fig.
6). Comparisons of the chromatograms, and the peak area or heights
between the drug and the blanks are made to demonstrate selectivity.
Blank chromatograms should be obtained from sample matrix (e.g.,
plasma) obtained from six different sources that have not been treated
with the drug. Furthermore, it is also advisable to determine whether any
medications to be co-administered during the clinical study will interfere
with the quantification of the analyte of interest. In addition, if an internal
standard is used in the method, blanks with internal standard should also
be compared to the drug and completely blank matrix to demonstrate that
the internal standard will not interfere with analyte quantification. For
other nonchromatographic types of analytical methods, such as RIAs and

FIGURE 6 Selectivity. The upper curve is a HPLC chromatogram of blank plasma.

In the middle tracing, drug X and an internal standard (ISTD) were spiked into
plasma. In comparison with the blank plasma, it can be concluded that the assay
provides good selectivity for this drug. The bottom chromatogram is an example of
assay in which the peak of interest (retention time of 10 min) is interfered with by a
larger unknown peak.

176 Booth and Simon
ELISAs, the demonstration of selectivity is more difficult because there is

no visual representation of the assay. In ligand-binding assays, an
antibody binds to some chemical entity, and quantification is based on
some radioactive tracer or enzyme activity. However, how does one know
that the antibody does not bind some entity other than the analyte of
interest? In these cases, the best assessment of selectivity is made by
screening ligand crossreactivity with other compounds known to be
chemically similar to the drug (i.e., endogenous compounds, drug
fragments, etc.). The difficulty is there may be interactions with
compounds that are not predictable. Therefore, the selectivity cannot be
known absolutely with these methods. In these cases, it also recommended
that selectivity of the ligand-based assays should be confirmed with the use
of other analytical methods that rely on different principles (e.g., HPLC).
In addition, nonspecific binding of the ligand may occur, and the prozone
effect, i.e., nonspecific binding with buffer constituents, should also be
assessed regularly [11].
Accuracy
The determination of accuracy indicates how close the measured concentra-
tion is to the true or nominal concentration (see Table 1). This step assesses
the systematic error or bias of the entire analytical procedure (analyte
extraction, reconstitution, analysis). Known amounts of analyte are added
to the matrix and measured. A minimum of three concentrations that span
the standard curve should be assessed, and at least five determinations or
replicates should be conducted for each concentration. Accuracy is
TABLE 1 Intra- and Between-Run Accuracy and Precision of Drug X

calculated as
The acceptance criteria for accuracy is ±15% of the nominal concentration,

but at the LLOQ an error of ±20% is permissible.
Precision
Precision is the determination of how close the repeated measurements of
the same concentration are to one another. A minimum of three analyte
concentrations that span the standard curve should be assessed, and at least
five determinations or replicates should be conducted for each
concentration. Precision is calculated as the coefficient of variation (% CV)
following repeated measurements.
Precision (% CV)=(standard deviation/mean) • 100 (see Table 1)
The acceptance criteria for precision is a coefficient of variation of ±15%,

but at the LLOQ a precision of ±20% is permissible. For the determination
of both accuracy and precision, within-day (within-run) and between-day
(between-run) determinations are made.
Recovery
Recovery is a measure of the ability of the extraction procedure to recover
the drug spiked into the biological matrix. Recovery is determined by
comparing the response of the analytical system to the analyte sample that
was extracted according to the analytical method, with the detector
response obtained from the same amount of pure authentic standard. The
recovery of the analyte does not need to be 100%, nor is it a required
element of method validation because, problems with recovery will be
detected by unacceptable measures of accuracy and/or precision. However,
during method development it is advisable to determine recovery in order to
diagnose problems with the analytical assay which may occur. Furthermore,
it is also advisable to determine the recovery of the internal standard
independently, if one is used.

178 Booth and Simon
Assessing the Effects of Sample Handling on Analyte

Stability and Quantification
The determination of sample stability indicates the extent of drug or
metabolite degradation that could be expected to occur as a result of sample
handling. In extreme cases of degradation, this information could prompt
the development of new sample-handling procedures. This is an important,
although frequently under-appreciated characteristic of an analytical
method. Typically, blood samples are collected according to a scheme
similar to the following
1. Blood sample withdrawal at the study site; blood samples may

be stored in ice for short periods.
2. Isolation of plasma from blood sample by centrifugation; this
operation may take 10 to 20 minutes, and the centrifugation
may or may not be refrigerated.
3. Plasma samples are then frozen and stored for some period.
4. Frozen plasma samples are transported to the analytical site;
commercial carriers are usually employed for transportion and
the samples are usually shipped on dry ice. The temperature at
which the samples are shipped may differ from the storage
temperature at the study site.
5. Plasma samples may be frozen at the analytical site for some
period before analysis; storage temperatures at the analytical site
may be different than those used at the study site or during
transportation.
6. The plasma samples are thawed, and aliquots of the sample are
processed and analyzed. The remaining plasma samples are
refrozen. These remaining samples may be rethawed and
reanalyzed at a later date.
This example illustrates that there are numerous opportunities for sample
degradation that could ultimately lead to erroneous pharmacokinetic
interpretations. Therefore, the chemical characteristics of the analyte should
be considered during the development of standard operating procedures
(SOPs) for sample collection. For example, the collection of samples for a
pharmacokinetic study of nitroglycerin is quite challenging. The elimination
half-life (t1/2) of nitroglycerin is two minutes in vivo, and once a sample is
withdrawn, the t1/2 in blood is six minutes. Therefore, it is imperative that
the plasma from these samples are isolated rapidly, under refrigerated
conditions, and frozen immediately.
Another consideration that should be borne in mind is that stability
testing should mimic the conditions of sample handling and storage to be
used in the study. There have been examples in which long-term stability

studies were conducted on samples stored at -70 °C. However, according to

the SOPs established in the study protocol, the samples were stored at -20
°C in practice, and the results of the stability study were of limited value
because the extent of sample degradation under the actual conditions of use
were not assessed. The assessment of analyte stability should be addressed in
the following discrete steps.
Freeze-thaw Stability
During the average study, it is likely that the samples may experience several
freeze-thaw cycles, and it is important to know the sensitivity of the analyte
to degradation resulting from this type of handling. This effect is assessed by
assaying spiked samples after three freeze-thaw cycles. Study samples
should be frozen for a minimum of 24 hours (at the temperatures planned
for storage in the clinical study), then thawed at room temperature. Once
completely thawed, the samples should be refrozen for a period of 12–24
hours. This cycle should then be repeated twice or more, and then the
samples should be analyzed. Low and high concentrations of the drug
should be assessed in triplicate.
Short-Term Room Temperature Stability

This characterization is meant to assess any degradation that may occur as
the samples are maintained on the benchtop prior to and during sample
processing (i.e., extraction, etc). Low and high concentrations of drug in
triplicate should be maintained at room temperature for the period of time
required for sample preparation and then analyzed.
Long-Term Stability
In this case, stability of the samples should be assessed according to the
planned storage conditions (e.g., -70°C), but for periods that exceed the
planned duration of storage. Three aliquots of low and high concentrations
need to be assessed three times during the planned period of storage, and
compared to the mean back-calculated concentrations of the sample
determined on the first day of the study. Care should be taken to make samples
with the necessary volume for repeated analyses. Interestingly, the Code of
Federal Regulations stipulates that sufficient quantities of samples must be
collected during a bioavailability (21 CFR 320.38) (11) or bioequivalence
(21 CFR 320.63) [12] study and stored for five years from the date of NDA
or ANDA submission. This regulation implies that longterm stability testing
of the analyte should span this period as well. However, this may be practically
impossible to achieve, and FDA does not require this step.

180 Booth and Simon
Stock Solution Stability

The stability of the stock solution of the analyte which would be used to
construct standard curves and quality control samples should be assessed
following approximately six hours at room temperature, and following
periods of refrigerated storage that are anticipated to be used during the
study.
Post-Preparative Stability
This characteristic may also be referred to as autosampler stability. The
stability of the processed samples should be assessed over the course of
analysis (i.e., the run time) according to the conditions of use (e.g., room
temperature or refrigerated autosampler). The stability assessments
described above should also be performed for any internal standard or drug
metabolites that may be measured in the assay, as well as the analyte of
interest.
Monitoring Assay Quality During Practical Use

Once the method has been established and validated, it is ready for
analytical use. However, as most current analytical methodologies employ
automation to increase productivity, analytical runs have become very long
(up to days). Therefore it is necessary ensure that the assay continues to
perform according to the specifications determined during the validation
stage throughout each analytical run. This is accomplished by making and
including quality control samples or calibrators (QC) of known
concentrations that can be interspersed with the calibration standards and
the clinical samples in each analytical run. The QC samples allow the
analyst to monitor the accuracy and precision of the method while it is in
use. QC samples are standards that are made of known quantities of drug
that is spiked into naïve matrix. A minimum of three concentrations that
bracket the standard curve should be prepared. The first QC sample should
be within 3×of LLOQ, the second QC sample should be mid-range and the
third QC sample at the upper end of the standard curve should be included.
The QC samples should be run in replicate. The QC samples should be
interspersed with the clinical samples and the standard curve calibrators,
but there is no consensus on how frequently QC samples should be
incorporated. The FDA recommends that 5% of the samples in the run
should be QC samples, but six QC samples are the absolute minimum for
any run. Both standard calibrators and QC samples should be arranged to
detect assay drift. In order to accept the analytical run, two-thirds of the QC
samples must be within 15% of their nominal values. For example, if six QC

samples were analyzed (two low QCs, two mid-QCs, and two high QCs)
and one replicate each at the mid- and high QC concentrations were greater
or less than 15% of nominal, the run would be deemed acceptable.
However, if two replicates at the same QC concentration failed (e.g., both
mid-QC samples in the example above), or more than two QC samples
failed, the analytical run would be rejected.
In addition to monitoring the method performance, it is also good
practice to include QC samples with the samples during storage. QC
samples can be prepared at the same time the samples are processed, and
stored with the samples to monitor storage conditions. This practice is
useful to guard against unforeseeable events, such as a power outage that
affects freezer function. This use of QC samples, although advisable, is not a
requirement of analytical method validation.
ANALYTICAL METHOD VALIDATION DATA FOR SUBMISSION TO FDA
Information that should be submitted in an NDA or an ANDA for the

analytical method validation should include the following:
• Summaries: A summary table that lists the validation studies by

title and number, and a table of the assay methods used in the
study (s).
• Method Establishment information: This should include a
description of the analytical method(s), evidence of analyte purity,
description of stability studies, description and tabulation of
accuracy and precision determinations, cross-validation studies
if necessary, legible chromatograms, or mass spectrograms
including blanks (up to 20% of chromatograms from three serial
patients for pivotal bioequivalence studies), and a list of deviations
from protocols and explanations for these deviations.
• Application of the validated method: Summary table of sample
handling, summary table of clinical or preclinical samples,
equations used, table of calibration curve data, summary tables
of intra and inter assay accuracy and precision, and of QC samples,
reasons for missing samples, reanalyzed samples, and reintegrated
samples.
PARTIAL VALIDATIONS AND CROSS-VALIDATIONS
The steps described above detail the process of complete or full validation
that is necessary for the development of a new analytical method. However,

182 Booth and Simon
there are two other method validation situations that require some
discussion. These situations are partial validations and cross validations.
Partial Validations
Periodically changes to a validated assay are necessitated for a variety of
reasons. For instance, due to protein binding, it may be necessary to switch
from heparin as an anticoagulant to EDTA. This apparently small change to
the validated assay may alter its performance and it is necessary to
demonstrate whether or not the characteristics of the assay have changed. A
full validation is likely not necessary, as a partial validation will suffice to
address the question. Unfortunately, the extent of partial validation is left to
the discretion of the analyst. Partial validations may range from one
intraassay accuracy and precision determination, to almost a complete
validation. A reasonable suggestion is that partial validations should
basically consist of selectivity, accuracy, and precision determinations. Once
this step is completed, the analyst may decide on the need for further
validation of the modified assay. Some of the situations where partial
validations should be considered are listed in the FDA Guidance. This list is
not exhaustive, but it describes the most likely partial validation situations.
Some of these scenarios are:
• Method transfer between labs or analysts

• Change in detection system
• Change in anticoagulants
• Change within matrix within species (e.g., human plasma to
human urine)
• Change of species within matrix (e.g., rat plasma to mouse
plasma)
• Changes in sample processing
• Change in concentration range
• Instrument or platform changes
• Limited sample volumes
• Rare matrices
• Selectivity demonstration of analyte in presence of concomitant
medications or in the presence of metabolites
Cross Validation
Cross validation of analytical methods is a special case. Cross validations
are a comparison of the validation parameters of two or more bioanalytical
methods. Generally, most bioanalysts develop and validate an analytical
method prior to the start of a clinical study. However, there are two

situations that can arise where cross validations should be conducted: when
two or more analytical methods are used to generate data within a single
study (including situations where one method was significantly changed
during the study), or when two or more analytical laboratories are used to
generate data within a single study. In addition, the analyst should consider
cross validation in cases where significantly different analytical methods
were used to generate data in different studies, if both studies produced data
of pivotal importance to the NDA. Unfortunately, there is no uniformly
accepted format for conducting cross validations. However, there are two
general approaches, which are quite similar. First, spiked samples of low,
medium, and high concentrations are simply analyzed by both methods and
compared.
Alternatively, clinical samples are analyzed by the different meth-
odologies and plotted against each other (see Fig. 7). Both methods should
provide the same value, and the slope of the line should equal unity. This
approach also allows certain statistical comparisons to be made [13].
Generally, the FDA recommends that both spiked samples and patients
samples should be compared between methods. However, it is also unlikely
that both methods will be exactly equal. The question then is how much
difference is acceptable. This issue has not been fully addressed, but usually
the ± 15/20% criteria used for accuracy and precision has been applied. It is
FIGURE 7 Cross validation. A set of patient samples were analyzed with two
different methods, A and B. The concentrations determined by each method are
plotted against one another. Ideally, if both methods were equal, they would
produce the same concentrations and a slope equal to one. In this case the slope is
0.66, which indicates that Method A reports higher concentrations than Method B.

184 Booth and Simon
advisable that the bioanalyst assess the objectives of the clinical study, and
set the requirements for cross validation appropriately.
CONCLUSIONS
The guidelines set forth in the FDA Guidance provide the framework that
can be applied to most cases of analytical method validation, regardless of
the analytical principle employed, and is most likely to assure the necessary
reliability of an analytical method. However, it is understood that there are
situations and methodologies where a validation cannot produce the degree
of accuracy or precision described. The over-riding question that needs to be
addressed by the bioanalyst is whether the analytical method reliably meets
the need(s) of the clinical study. In these cases, if the bioanalyst has
demonstrated due diligence and effort in method development, and the
reliability of assay given the requirements of the study, validations with
lower standards may also be deemed acceptable.
REGULATORY WEBPAGES
Australia, Therapeutic Goods Administration: www.health.gov.au/tga/

Canada, Therapeutic Products Directorate: www.hc-sc.gc.ca/hpfb-dgpsa/
Europe,EMEA:eudraportal.eudra.org/
International Committee on Harmonization: www.ifpma.org/ichl.html
Japan, Ministry of Health and Welfare: www.mhw.go.jp/english/index.html
U.S. FDA: www.fda.gov/cder/guidance/index.htm
REFERENCES
1. Guidance for Industry: Bioanalytical Method Validation 2001. www.fda.gov/

cder/guidance/index.htm
2. Shah, V.P.; Midha, K.K.; Dighe, S.; McGilveray, J.J.; Skelly, J.P.; Yacobi, A.;
Layloff, T.; Viswanathan, C.T.; Cook, C.E.; McDowell, R.D.; Pittman, K.A.;
Spector, S. Analytical Methods Validation: Bioavailability, Bioequivalence and
Pharmacokinetic Studies. Pharm. Res. 1992, 9, 588–592.
3. Guidance for Industry: Bioanalytical Method Validation in Human Studies
Posted in 1999. www.fda.gov/cder/guidance/index.htm
4. Shah, V.P.; Midha, K.K.; Findlay, J.W. A.; Hill, H.M.; Hulse, J.D.; MacGilveray,
I.J.; McKay, G.; Miller, K.J.; Patnaik, R.N.; Powell, M.L.; Tonelli, A.;
Viswanathan, C.T.; Yacobi, A. Workshop/Conference Report Bioanalytical
Method Validation—a Revisit with a Decade of Progress Pharm Res 2000, 17,
1551–1557.

5. Guidance for Industry: Analytical Procedures and Methods Validation

Chemistry, Manufacturing and Controls Documentation, www.fda.gov/cder/
guidance/index.htm
6. Reviewer Guidance: Validation of Chromatographic Methods, www.fda.gov/
7. Guideline for Submitting Samples and Analytical Data for Methods Valida-tion.
www.fda.gov/cder/guidance/index.htm
8. Jennings, W. Analytical Gas Chromatograpgy. Academic Press: San Diego,
1987; pp. 1–23.
9. Causon, R. Validation of Chromatographic Methods in Biomedical Analysis:
Viewpoint and Discussion. J. Chromatog. B 1997, 689, 175–180.
10. ICH Topic Q2B: Validation of Analytical Procedures: Methodology,
www.eudra.org/emea.html
11. Oldfield, P.R.; Pham, K.; Ng, A. The Effect of Prozone on Toxicokinetic Data—
a Case Study. American Association of Pharmaceutical Scientists Annual
Meeting, 2000 Abstract 3182.
12. Code of Federal Regulations, Title 21 parts 320, 2000, 185–199.
13. Gilbert, M.T.; Barinov-Colligon, I.; Miksic, J.R. J. Pharm. Biomed. Analysis
1995, 13, 385–394.

9
Studies of the Basic Pharmacokinetic
Properties of a Drug—a Regulatory
Perspective
Maria Sunzel*
INTRODUCTION
This chapter concerns basic pharmacokinetic studies that are essential for
understanding the characteristics of a new chemical entity; however, all
types of studies are not covered by specific regulatory guidance documents
or regulations. The majority of these studies are performed early in the
clinical development of a new chemical entity. Single-dose studies form the
basis of the pharmacokinetic knowledge needed for a rational drug
development program. Repeated-dose studies confirm results obtained after
single-dose administration, but can also reveal time-dependencies,
nonlinearity, and self-induction/inhibition in the pharmacokinetics of a
drug. If adequate information is captured early in development, the need for
Current affiliation: AstraZeneca LP, Wilmington, Delaware, U.S.A.
187
188 Sunzel
additional Phase I studies, e.g., to elucidate apparent inconsistencies in basic

pharmacokinetic properties observed in early studies, may be reduced, and
the appropriate designs of early Phase II studies can be selected with added
confidence. If essential pharmacokinetic knowledge is obtained early on in
the development, a potentially negative result of an early proof-of-concept
study in the target (patient) population would more likely reflect drug
effects rather than a miscalculation of the dosage regimen. It is desirable that
the drug levels should be monitored in such a proof-of-concept study, to get
insight and knowledge of preliminary exposure (pharmacokinetic)-response
(pharmacodynamic) relationships of the drug. It is also advisable to
investigate potential exposure-response relationships throughout all stages
of drug development. Readers are referred to Chapters 10 and 11 for a more
detailed description of such studies.
The studies that will be discussed in this chapter are early safety and
tolerability studies, mass balance or ADME studies, dose proportionality
studies, bioavailability studies, food interaction studies, and repeated dose
studies. In the review of a new drug application (NDA), evaluation of the
validation of the bioanalytical methods such as specificity, sensitivity, limits
of detection, and quantitation plays an important role in the overall
assessment of the validity of the pharmacokinetic data. Chapter 8 describes
the analytical method validations that should be performed prior to
conducting these studies.
The Guidance documents issued by the U.S. Food and Drug
Administration (FDA) referred to in this chapter can be found on the FDA’s
website www.fda.gov/cder. A summary of the Code of Federal Regulations
(CFRs) quoted in this chapter can be found in Chapter 3, or in the Federal
Register. For specific regulations by other regulatory agencies in the world,
readers are referred to the specific agency’s website and encouraged to
contact the appropriate agency for additional information they may need.
SINGLE-DOSE STUDIES
The major part of the basic properties of a chemical entity can be

extrapolated from single-dose studies if the pharmacokinetics of the drug
are linear. Linear pharmacokinetics is described by an increase in dose that is
followed by a proportional increase in exposure of the drug (e.g., the area
under the plasma concentration-time curve), over the anticipated
therapeutic dose interval. The basic pharmacokinetic parameters of a drug
from the single-dose studies can then be used for predictions of drug
exposure after repeated doses, after various dosing regimens [1].
Indications of nonlinear pharmacokinetics should be investigated early
to determine if the cause is related to absorption, distribution,

Basic Pharmacokinetic Properties of a Drug 189
metabolism, or excretion processes. It is generally recommended that the

pharmacokinetic studies are performed in fasting subjects (overnight fast),
to reduce the influence of potentially confounding factors elicited by
concomitant food intake. On the other hand, it is most desirable that
potential influence of food on the pharmacokinetics of the drug also is
investigated early on in the drug development program. This information
facilitates appropriate recommendations as how the drug should be
administered in the Phase II or Phase III trials in the target patient
populations.
Safety and Tolerability

The initial study, where first dose is administered in humans, yields
valuable information regarding basic pharmacokinetic properties of a new
chemical entity, and can give indications about potential nonlinearities in
the pharmacokinetics. This safety and tolerability study is usually
conducted in healthy adult volunteers, where subjects are administered
escalating doses of the drug, starting from low doses that are increased in
a stepwise manner. Generally, safety parameters are intensively monitored,
and volunteers scheduled for the next dose level are not dosed until a
safety evaluation from the previous cohort of subjects is completed. The
maximum dose in the study is usually not predetermined, but is limited by
adverse events or by predetermined stopping rules. Recommendations of
the preclinical toxicological studies that should be completed and
evaluated before the first human trial is initiated are described in the ICH
Guidance “Non-clinical safety studies for the conduct of human clinical
trials for pharmaceuticals” [2].
Choice of Dose
The starting dose and the subsequent dose increments are generally chosen
according to the preclinical pharmacological and toxicological results.
The less toxic effects a drug has shown to produce, the larger dose
increments can be made, at least during the initial part of the dose-
escalating trial. Criteria for stopping rules of the dose-escalation, i.e., the
maximal dose given in the study, should be predetermined and specified in
the protocol, as far as possible. The stopping rules may include a number
of subjects that experience moderate to severe adverse events, plasma
levels where preclinical toxicological findings limit further dose increases,
or established surrogate maximal endpoints that have been reached. The
first safety and tolerability study can provide considerable insight
regarding the therapeutic index of a drug if an adequate dose range is
explored.

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Assessments of the exposure-response relationships for a new chemical

entity in a preclinical animal model may give sound directions for the
therapeutic concentration (exposure)-effect (response) relationships to be
evaluated in the first safety and tolerability study, as well as subsequent
studies. Although surrogate endpoints or biochemical markers usually are
used as an alternative to the clinical endpoints used in the later confirmatory
Phase III trials, early information regarding exposureresponse correlations
from both preclinical animal and healthy volunteer studies could aid further
drug development. Naturally, the chosen surrogate endpoints or markers
should capture information that is considered to be applicable to the future
patient therapy. The exposure-response relation-ships determined in the
preclinical pharmacological and toxicological studies can also guide the
magnitude of dose escalation steps in the first study. A steep exposure-
response correlation calls for smaller dose increases compared to a more
shallow correlation between dose or concentration and pharmacological or
toxic effects of the drug. However, the assumption is that the metabolism
and activity of the drug and metabolites are similar in the animal species and
humans. For example, a particular metabolite contributing towards
toxicological or pharmacological effects may be formed in humans but not
in animals, which may, in part, invalidate predictions based on preclinical
observations.
Interspecies scaling is used as an instrument to predict pharmacokinetic
parameters and exposure in humans. Two techniques, physiologic and
allometric scaling, and more recently, allometric scaling in combination
with in vitro-in vivo correlations, are extensively described in the literature
[3–6]. Interspecies scaling techniques are also described in detail in Chapter
7. The allometric scaling approach may be very useful as an aid for
predictions of the dose interval to be investigated in the first safety and
tolerability study. At present, there are no requirements or final guidance
documents regarding the use of scaling techniques for dose selection in
Phase I studies. However, the FDA has recently published a draft Guidance
[7], which mainly focuses on an algorithm for calculations of the maximum
recommended starting dose (MRSD) in humans from animal data. The
described algorithm for these estimations include appropriate safety
margins for the MRSD is based on available no observed effect levels
(NOEL) in animals. Allometric scaling and modeling are also considered,
and it is recommended that an adequate safety factor for the MRSD is also
included, if such approaches are chosen.
A combination of allometric scaling techniques and knowledge of the
exposure-response relationships has indeed proved to be worthwhile. In a
survey from one major pharmaceutical company it was estimated that
timesavings of two weeks to six months could be accomplished in the first
safety and tolerability study by utilizing exposure-response correlations and

allometric scaling techniques from preclinical studies [8]. The major

advantage was a reduction of dose steps in the low, subtherapeutic dose
range.
Study Population
The study population in the first safety and tolerability study is usually
healthy, adult male and female volunteers aged 18–45 years old, with
normal weight in proportion to their height. Since the preclinical
reproduction toxicity studies may not have been completed when the first
human safety study is performed, women of childbearing potential may be
excluded from that study population. However, it is highly recommended
that women are included as early as possible in the first human clinical
pharmacology studies [9–11].
As a matter of fact, as stated in the ICH Guidance document ICH M3 [2],
there are regional differences across the world in the recommended timing
of reproduction toxicity studies to support the inclusion of women of
childbearing potential into human trials. The regional differences outlined
in ICH M3 are as follows:
• The United States: Women of childbearing potential may be

included into carefully monitored trials before the reproduction
toxicity studies have been completed. Recommended safety
measures include pregnancy testing, the uses of a method of birth
control considered as highly effective, and study entry after a
verified menstrual period.
• The European Union: The evaluation of embryo-fetal
development should be completed prior to Phase I trials, and
female fertility before Phase III trials are initiated, in women of
childbearing potential.
• Japan: Assessment of female fertility and embryo-fetal
development should be completed before women using birth
control are included in any type of trial. Permanently sterilized or
postmenopausal women may be included into trials before
reproduction toxicity studies have been completed, if the
appropriate repeated toxicity studies have been performed,
where any toxicity related to the female reproductive organs
have been evaluated. A male fertility trial should be completed
before the Phase III trials are started.
If the target patient population only encompasses a certain specific

population, e.g., women for oral contraceptives or hormone-replacement
therapy, or drugs for Alzheimer’s disease in the elderly, more adequate

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information could be gathered by performing the early Phase I studies in the

intended target population (e.g., women or elderly subjects). In certain cases
when the toxicity of the drug is expected to be high, e.g., drugs intended for
treatment of cancer, it might be unethical to perform any trials in healthy
volunteers, thereby exposing healthy subjects to drugs that may cause undue
harm. All these factors should be considered at the time of design of these
studies.
Study Design
The first safety and tolerability study in humans is usually performed in
single escalating dose, open, or single-blind, parallel design. The number
of subjects included in each dose level is generally limited (n=3–8), where
the number of subjects is increased at higher dose levels. A parallel
design is usually chosen to increase the number of subjects that are
exposed to the drug, thereby maximize early safety information
regarding the pharmacological or toxicological effects on variables such
as vital signs, clinical chemistry, and adverse events. A parallel group
design may also reduce the risk for the individual volunteer if unexpected
adverse events occur where repeated exposures may augment the
unforeseen adverse events. A limited placebo control group can also be
valuable, especially if the pharmaceutical formulation contains an
excipient or a vehicle that may elicit a pharmacological or a
toxicological response.
An adequate number of blood samples is recommended to ensure, as far
as possible, that a full plasma concentration-time profile is attained.
Data Analysis
Accurate information regarding the maximum drug plasma concentration
(Cmax), area under the plasma concentration-time curve (AUC), terminal
half-life (t½) of the drug, and the interindividual variability are valuable for
future study designs. The methods for calculation of the parameters are
discussed in the section “Data Analysis” on page 199 of this chapter.
Although the number of subjects usually is limited in the first human
study, initial information regarding dose linearity, i.e., proportional
increases in exposure (Cmax and/or AUC) with increasing doses, can be
made. An attempt to evaluate information regarding relationships
between plasma concentrations of drug and pharmacological effects,
surrogate markers, or adverse events is also valuable. Any information
regarding such relationships would enhance appropriate future study
designs.

ADME (mass balance)
The absorption-distribution-metabolism-elimination (ADME) study in

humans is not only one of the most informative, but also one of the most
labor intensive, Phase I studies. Although in vitro studies yield qualitative
information regarding metabolism across species, quantitative information
can only be obtained from in vivo studies. The timing of the ADME study in
relation to other studies in the clinical development program varies.
However, the earlier the study is performed, the more useful are the results
from the study. Early information regarding major metabolites and
excretion patterns is essential for rational planning of studies, e.g., for
special populations. Since elucidation of metabolic patterns may be
timeconsuming, it is advantageous to initiate the ADME study as one of the
first Phase I studies. It is obvious however, that the choice of dose and
sampling collection at appropriate time intervals is essential for a good
outcome of the study, therefore knowledge about the basic pharmacokinetic
properties of the drug should be attained before the ADME study is
initiated.
Choice of Dose
The dose of the radiolabeled drug should be kept as low as possible.
Information regarding tissue distribution in animals, e.g., from whole body
autoradiography studies, provides valuable information about high drug
accumulation in specific tissues, as well as the time course of elimination
from specific tissues. The information can also be utilized in the risk
assessment of the use of radioactive isotopes for human studies. The
regulations regarding the use of isotopes in human research vary between
different countries. Dosimetry calculations to estimate exposure in different
tissues need to be performed, and in general the protocol has to be approved
by a Radioactive Drug Research Committee as well as an Investigational
Research Committee. In the United States, the rules for the use of
radiolabeled drugs in research can be found in 21 CFR 361.1, and the reader
is also referred to a related overview by Dain et al. [12].
The choice of radiolabel for the drug is usually dependent on the isotope
that was chosen for the mass balance studies in the animal species. The same
isotope should be used in the human in vivo study to enable crossspecies
comparisons of metabolic patterns. This is important, since the metabolic
pattern should be similar between the animal species chosen for the
preclinical carcinogenicity and long-term toxicity studies and humans. If the
metabolic profiles differ substantially between humans and animals,
additional (preclinical) studies may be needed. For example, if a major
metabolite is formed in humans, which has not been observed in animal

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studies, then this metabolite may have to be synthesized and administered to

animals to assess the pharmacological and toxicological properties of the
particular metabolite. In such cases, the appropriate regulatory agency
should be contacted to get their guidance on which additional studies may
be needed, or to discuss the adequacy of additional study protocol
proposals. The radiolabel should be properly positioned in the molecule to
yield relevant information regarding the drug metabolism. The
radiochemical purity is also important, especially for protein-binding
assessments of highly (>99%) protein-bound drugs [13].
Study Population
The ADME study is usually performed in healthy, adult, male volunteers,
18–45 years of age. Women are traditionally excluded due to the potential
risks associated by exposing females of childbearing potential to a yet
unapproved, radiolabeled drug. By the same token, certain investigators
limit the lower age limits of the male volunteers to an age arbitrarily chosen
above 18, for example an age of 35 years, and may extend the upper age
limit to 60 years. The number of subjects is usually low (n=4–8), but some
caution should be used in keeping the number of subjects high enough, so
that the results will be informative. If the drug has shown highly variable
pharmacokinetics in earlier studies, a larger number of subjects may have to
be included in the study.
Study Design
The optimal design of an ADME study is a crossover, or a parallel group,
study where an intravenous (IV) dose serves as a reference to the enteral
(e.g., oral, rectal, or sublingual) or other parenteral (e.g., topical or
pulmonary) routes of administration. Even if the development of the new
chemical entity is only focused on, e.g., an oral route of administration, the
pharmacokinetic information from an IV dose will significantly enhance the
understanding of the pharmacokinetics of the drug, especially information
regarding absorption processes, presystemic metabolism, and first-pass
effects. However, a study design, where only one route of administration is
chosen, would be satisfactory, although more limited information regarding
the ADME processes will be collected.
Blood and plasma samples, aliquots of urine and feces, and in certain
cases expiration air, are collected over an extended period of time. The time
period for collection of biological specimens is obviously governed by the
terminal half-life of the drug and/or metabolite(s), and can be determined by
“on the spot” quick-counts of radioactivity in, e.g., urine or feces. The
blood-sampling period is usually terminated well ahead of urine and feces

collection, where the latter usually continues for 7–10 terminal half-lives of
the drug or metabolite(s). It is essential that the recovery of the total
radioactivity in the different biological fluids is 85–90% or above, therefore
strict provisions regarding sampling collection need to be made. The
volunteers need to be fully informed and understand the importance of
complete collection of urine and feces specimens, and comply with the
instructions.
The metabolite identification is performed in the biological samples after
extraction and separation (e.g., by fractional collection). Metabolite
identification should be attempted in all the collected biological specimens
(e.g., blood or plasma, urine, feces). The metabolite structures are generally
identified by use of liquid chromatography-(tandem) mass spectrometry
methods [14]. Accelerator mass spectrometry (ACL), which has been used
for areas such as age determination of archeological objects, has recently
been applied in biomedical research, e.g., ADME studies [15, 16]. The main
advantage with this technique is a very high sensitivity and precision, which
permits the use of extremely low doses of radiolabeled materials and
quantitation of low levels of radioactivity. However, this promising technique
is not yet used routinely, and may require further validation. All analytical
methods need to be adequately assessed, as described in Chapter 8.
Data Analysis
The data analysis is usually extensive. Graphs of the time-course of
excretion (e.g., urine and feces) and plasma/blood profiles of total
radioactivity, as well as of each analyte should be constructed. The ratio of
parent compound and each metabolite to total radioactivity may also be
calculated.
Pharmacokinetic parameters, e.g., AUC, Cmax, tmax, total clearance (CL),
renal CL, terminal half-life, apparent volume(s) of distribution (Vγ), and
amount of drug excreted unchanged in urine (Ae), should be calculated for
the drug. The corresponding parameters should, if possible, be calculated
for the major metabolite(s). If an IV dose is administered, absolute
bioavailability and actual CL and Vγ values can be calculated. An IV dose
can be extremely valuable, since any quantitative differences in metabolism,
excretion patterns and CL between IV and oral administration, as well as a
measure of the absolute bioavailability and extraction ratio, will aid the
understanding of the disposition of the drug. Incomplete absorption can be
detected from differences in excretion patterns and presystemic metabolism
can be detected from different metabolite/parent ratios between different
routes of administration. The report is enhanced when it contains clear
graphs and tables of both individual and average data, as well as summary
statistics. Due to the exploratory nature of the ADME study only descriptive

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statistics are expected. If the information is available, a scheme of the

proposed metabolic pathways in humans adds valuable information to the
study report.
Bioavailability
Definitions
Absorption of the active moiety is a stipulation for systemically acting drugs

that are administered by an extravascular route [1]. Bioavailability is
defined as the rate and extent of absorption of the intact drug or active
moiety. Studies that concern the evaluation of dose-linearity, potential food-
drug interactions, and the pharmacokinetics after repeated administration
are discussed in subsequent sections of this chapter. Alternative approaches,
i.e., pharmacodynamic studies, to those described in this chapter might be
necessary for locally acting drugs, where systemic exposure is not intended
and cannot be assessed. However, if the bioavailability (or bioequivalence)
of a drug can be determined by a pharmacokinetic study, a
pharmacodynamic approach is not recommended.
Bioavailability and especially bioequivalence studies are generally
performed throughout a product’s life cycle, both before and after the drug
approval. Bioequivalence studies are the principal basis for approval of
abbreviated NDAs for generic drugs. These studies are essential for both
efficacy and safety, by demonstrating that the pharmaceutical formulation
gives reproducible drug exposure, and intended plasma levels of the active
moiety. Bioequivalence studies are discussed in detail in the
Biopharmaceutics section, and will not be discussed in this chapter.
The European Agency for the Evaluation of Medicinal Products (EMEA)
has issued a new guidance document regarding investigations of
bioavailability and bioequivalence in July 2001 [17]. In the United States,
the requirements for bioavailability and bioequivalence studies for product
approval are described by the Code of Federal Regulations (21 CFR 320),
and more details are found in Chapter 2. In 21 CFR 320.1, bioavailability is
defined as “the rate and extent to which the active ingredient or active
moiety is absorbed from a drug product and becomes available at the site of
action. For drug products that are not intended -to be absorbed into the
bloodstream, bioavailability may be assessed by measurements intended to
reflect the rate and extent to which the active ingredient or active moiety
becomes available at the site of action.”
As an additional support for adequate designs of bioavailability (and
bioequivalence) studies, FDA has published several guidance documents
regarding the general principles for these studies:

• “Bioavailability and Bioequivalence Studies for Orally

Administered Drug Products—General Considerations”
(Revision 1, March 2003)
• “Food-Effect Bioavailability and Fed Bioequivalence Studies”
(December 2002)
• “Statistical Approaches to Establishing Bioequivalence”
(January 2001)
• “Extended Release Oral Dosage Forms: Development,
Evaluation, and Application of In Vitro/In Vivo Correlations”
(September 1997)
• “Waiver of In Vivo Bioavailability and Bioequivalence Studies
for Immediate-Release Solid Oral Dosage Forms Based on a
Biopharmaceutics Classification System” (August 2000)
The guidance documents relating to bioequivalence and conditions where

waivers are granted in lieu of in vivo studies are discussed in detail in the
chapters in the Biopharmaceutics section of this book. It should be noted
that the guidance documents are recommendations, and reflects the current
thinking of the FDA. Alternative approaches than those recommended in
the guidance documents may be employed if the requirements of the statutes
in 21 CFR 320 are fulfilled.
Methods
The most commonly used method to determine the rate of absorption is by
reporting the time (tmax) to reach the (observed) peak plasma concentration
(Cmax) of drug after dose intake. The observed Cmax of the administered drug
characterizes the peak exposure after dose intake. Other methods to
determine the rate of absorption may be employed, which may be more
meaningful for the comprehension of the absorption processes of the drug,
since tmax and Cmax are governed by both absorption and elimination
processes. Examples of other methods are deconvolution or calculations of
the absorption rate constant (ka), and can also be utilized [18].
The extent or completeness of absorption of intact drug or the active
moiety is usually expressed by the area under the plasma concentration-time
curve, AUC, as a quantitation of exposure. Comparative bioavailability is
expressed as a fraction (or percent) of the administered dose, where another
pharmaceutical formulation or route of administration serves as reference.
Comparative bioavailability (F) is calculated as:

198 Sunzel
where AUC denotes the area under the plasma concentration-time curve,
and dose adjustments are performed if unequal doses of the test and
reference drugs are administered. Alternative biological fluids, e.g., whole
blood or urine, can also be used for the determination of bioavailability.
Absolute bioavailability (F) is determined after administration of an
intravenous reference dose, where the intravenously administered dose is
assumed to be 100% bioavailable. Relative bioavailability (F rel) is
determined when the reference dose is administered extravascularly, e.g., as
an oral solution or a suspension. Early indications of a lower Frel than
expected may call for additional modifications of the drug substance where
ultra micronization or other measures may increase the in vivo absorption
of the drug.
In certain cases, absorption is the slowest, rate-limiting step in the
disposition of a drug. Differences in terminal t1/2 of the drug after different
routes of administration may indicate rate-limiting absorption processes [1].
Again, an intravenous reference dose is one of the most straightforward
ways to determine the basic pharmacokinetic properties of the drug or
formulation, since the intravenous route of administration circumvents all
absorption processes.
Relative or absolute bioavailability of the dosage form should to be
established. In early stages of drug development, the oral tablet
formulations are usually of immediate release (IR) character, and an oral
solution, or suspension, are used as the reference if an intravenous
formulation is not available. This study can be valuable as a point of
reference, if subsequent modifications and optimizations are made to the
dosage form during further drug development. It is possible to link
formulation changes by bioavailability studies between formulations, and in
vitro dissolution comparisons may also preclude in vivo studies if only
minor modifications are made. However, major changes between clinical
trial formulations and/or the formulation intended for commercial use may
warrant bioequivalence studies (see related chapters in the
Biopharmaceutics section).
Study Population
Bioavailability studies are usually performed in healthy, adult volunteers,
above 18 years of age. Inclusion of equal numbers of men and women, or
volunteers resembling the patient target population (e.g., elderly), is
encouraged. The number of subjects participating in the study should be
based on earlier studies where intersubject, and, if available, intrasubject
variabilities have been determined.

Study Design
A single-dose, randomized, crossover design is the most common choice for
a bioavailability study. Study drug should be administered with 240 mL (8
oz) of water after overnight fast and standardized meals should not be
served until four hours post-dose. Water ad lib is allowed ± 1 hour of dose
intake.
In rare cases, a parallel-group design may be selected instead of a
crossover design. Drugs with a long terminal half-life may preclude the
choice of a crossover design, due to practical aspects of sample collection.
For a comparative bioavailability study of a drug with a long terminal
halflife, an alternative design, e.g., the “semi-simultaneous” method, may
be considered. In the “semi-simultaneous” approach, the test and
reference doses are administered at one occasion, but the doses are
separated by a certain time interval and no washout period is employed
[19]. However, it is recommended that any nontraditional study design
should be discussed with the regulatory agencies prior to study initiation,
to determine the regulatory view on the appropriateness of the specific
design.
Blood samples should be collected to adequately describe the full
plasma/serum drug concentration profile, including absorption,
distribution, and elimination. It is essential to characterize the absorption
phase (predose and 1–3 samples before Cmax), as well as the terminal phase
(≥3 samples) of the plasma concentration-time profile, where sampling
should be continued up to at least three terminal t½ of the drug/active
moieties. Investigational periods should be separated by an adequate
washout interval (>5t½) to ensure that elimination is complete before the
second dose is administered.
Data Analysis
Standard pharmacokinetic parameters, area under the plasma
concentration-time curve (AUQt and AUC∞), observed maximum plasma
concentration (Cmax), time to maximum plasma concentration (t max),
elimination rate constant (γz), and terminal t½ are routinely calculated for
the intact drug as well as any active metabolites. AUQt is calculated from
time zero (time of dose intake) to time t, where t is the last time-point with a
measurable drug concentration (Ct) in plasma. AUQt is calculated by the
linear or log-linear trapezoidal method. AUC∞ is calculated from time zero
to infinity, where AUC∞=AUQt+Ct/γZ.
As stated earlier, other methods to determine the rate of absorption better
than tmax and Cmax may be employed. For regulatory purposes, however, the
observed Cmax and tmax should always be included in the data analysis and

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report. Compartmental methods may also be used for calculations of AUC,

but in general, noncompartmental methods, such as the trapezoidal method,
are preferred. As a matter of fact, the European Guidance [17] does not
recommend the sole use of compartmental calculation methods for the
analysis of bioavailability or bioequivalence studies.
For a comparative bioavailability study, 90% confidence intervals should
be constructed for the log-transformed ratios of AUCt, AUC∞, and Cmax for
the test and reference formulations. If unequal doses of test and reference
formulations are administered, dose corrections should be included in the
calculations. Although the objective of a comparative bioavailability study
differs from confirmatory bioequivalence studies, i.e., 80–125% as a pass
criterion does not have to be fulfilled, it is highly recommended that 90%
confidence intervals for ratios of AUCt, AUC∞, and Cmax for the test and
reference formulations be reported. The report should contain clear graphs
and tables of both individual and average data, as well as summary
statistics.
Food-Drug Interactions
Concomitant food and drug intake has the potential to cause altered drug
absorption due to physicochemical and/or physiological reasons [20]. The
absorption process is in part dependent on the physicochemical properties
of a drug, such as pKa, rate of dissolution, and chemical stability, which all
may be altered by concomitant food intake. Certain effects may readily be
predicted from the chemical properties of a molecule, e.g., an acid-labile
structure will be subject to an increased rate of degradation due to
prolonged residence time in the stomach, where absorption of the drug will
be decreased after concomitant food intake. A suitable pharmaceutical
formulation can prevent such a phenomenon by, for example, enteric
coating of the oral tablet to protect the drug substance to premature
degradation.
Food also alters gastrointestinal physiology compared to the fasting
state, by delaying gastric emptying, changing pH in parts of the
gastrointestinal tract and increasing visceral blood flow, among other
effects. All these changes may modify the absorption of the drug, but some
might also be quite easily predicted by examining the inherent chemical or
pharmacokinetic properties of the substance. The composition of the meal,
such as the fat, protein, and overall caloric content can also influence the
magnitude of an observed interaction. The FDA has recently published a
guidance document entitled “Food-Effect Bioavailability and Fed
Bioequivalence Studies” [20], which is available on the FDA’s website:
www.fda.gov/cder.

Choice of Dose and Composition of the Meal

A study investigating the potential influence of concomitant food intake
should be performed under conditions that really stresses the system, that is
a “worst case” approach should be used. Therefore, the highest dose in the
expected therapeutic range should be chosen. A sound justification for the
use of a lower dose strength is recommended, e.g., tolerability problems that
precludes dosing at the highest dose level without previous dose titration
starting at a lower level. If a modified release (MR) formulation has been
developed, in vitro dissolution testing can be substituted for an in vivo study
for other, usually the lower, strengths of the MR tablets. If the in vitro
release profiles between the MR formulations differ, or the excipients differ
qualitatively between the dosage strengths, additional in vivo food studies
may be required for the other dosage strengths.
The composition of the meal should be of high caloric content
(approximately 800–1000 calories) where 50% of the content consists of
fat. The FDA gives an example of test meal, which fulfills these criteria,
which is composed of two eggs fried in butter, two strips of bacon, two
buttered slices of toast, four ounces (about 110g) of hash brown potatoes,
and eight ounces (240 mL) of whole milk [20]. This meal gives about 150
calories from protein, 250 calories from carbohydrates, and 500–600
calories from fat. Alternate meal compositions can be used, but it is
important that the proportions of fat, protein, and carbohydrates are kept
to give a similar caloric content to the proposed test meal. The description of
the meal should be included in both the protocol and the final report.
One may argue that the described breakfast is not an appropriate test
meal for the vast majority of patients, since only a fraction of any
population eats this type of breakfast. However, the purpose of the test meal
is to study the effects of maximal perturbations created by concomitant
food intake, both with respect to interaction between the drug, the
pharmaceu-tical formulation, and the nutritional content of the meal. The
high caloric content, in part originating from the high fat content, will also
amplify the physiological effects of the test meal, e.g., the delay in gastric
emptying and the increase in splanchnic blood flow.
Study Population
As described in the previous section (Bioavailability) the study is usually
performed in healthy, adult male and female volunteers, above 18 years of
age, unless the study is conducted in the target patient population. It is
advisable to perform the, study in the target patient population if the
indication of the orally administered drug is to treat a disease likely to alter
drug absorption, e.g., inflammatory bowel disease. The sample size should

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be based on earlier determinations of intersubject variability, although it is

recommended that a minimum of 12 subjects is included in the study.
Study Design
The most commonly used study design is a balanced, randomized, two-way
crossover study, analogous to a bioavailability study, as described in Section
2.3.4 of this chapter. The subjects are given a single dose of the study drug in
the fasting state (reference) and after a meal (test). Both the treatments
should be preceded by an overnight fast (at least 10 hours), and the
treatments should be separated by an adequate washout period.
• Reference treatment (fasting state): The drug should be adminis-

tered with 240 mL (8 oz) of water. Water intake is permitted ad
lib, except within ± 1 hour of drug intake, but standardized
meals should not be served until four hours post-dose.
• Test treatment (fed state): The test meal should be consumed
within a prespecified time interval (30 min) and the study drug
should be administered with 240 mL (8 oz) of water immediately
after completion of the meal. Water intake is permitted ad lib,
except within ± 1 hour of drug intake, but standardized meals
should not be served until four hours post-dose.
Additional studies might be necessary if an undesired food-drug interaction

is observed which warrants special dosing recommendations regarding the
timing of the meal in relation to dose intake. Especially, if the
pharmacological effects are mainly related to peak concentrations rather
than total exposure of the drug, and concomitant food intake reduces the
Cmax of the drug, the optimal time interval between the meal and dose intake
should be explored to reduce the risk of therapeutic failure.
Data Analysis
Standard pharmacokinetic parameters, Cmax, tmax, lag time (for delayed
release products), AUQt, and AUC∞, should be calculated for the intact drug
and it is also valuable to calculate these parameters for major, active
metabolite(s). The terminal half-life should also be reported. The reader is
referred to the section “Data Analysis” on page 199 of this chapter, for a
more detailed description of the calculations. The report should contain
clear graphs and tables of both individual and average data, as well as
summary statistics. The evaluation of the absence or presence of a food
effect is based on the 90% confidence intervals (CI) for the ratio of the
means of the test (fed) and reference (fasting) conditions of Cmax and AUG.

Absence of a food effect is concluded when the 90% CI for the ratio of the
population geometric means (based on log-transformed data) met the limits
of 80–125% for AUC and Cmax.
If a food effect has been observed (>20% difference in AUC and Cmax
between fed and fasting states), the clinical relevance of this finding should
be considered in relation to the dose (or exposure)-response relationships of
the drug. The dosing recommendations should reflect the optimal timing of
food intake in relation to drug administration, so the intended therapeutic
effects of the drug are maintained. The clinical relevance of an observed
change in the rate of absorption (tmax or lag time) between the fed and fasted
states should also be considered and addressed in an NDA submission.
Regulations regarding labeling requirements in the United States can be
found in 21 CFR 201. The evidence of absence or documented food effects
should be stated in the product labeling for the drug, and the “Dosage and
Administration” section of the labeling should provide the instructions for
drug administration in relation to food.
Timing of the Study

The objective of an investigation regarding the influence of food intake can
be related to the drug substance in itself, or also be related to the
pharmaceutical formulation. Early identification of a food effect is of value
to optimize dosing recommendations in subsequent clinical trials or serve as
a basis for attempts to minimize influence of the food by modification of the
drug substance (e.g., micronization) or the pharmaceutical formulation.
From a regulatory perspective, the information regarding food effects in a
submission should be based on the to-be-marketed pharmaceutical
formulation.
For an IR formulation, a study that indicates a substantial food effect
performed early in development using a prototype IR formulation might not
need to be repeated at a later stage. However, such a conclusion needs to be
ascertained by reasonable information that shows that the food effect or
absence thereof is due to the drug substance and not the formulation or
processing factors. A food-effect study for a modified release (MR)
formulation should always be performed on the highest dose strength of the
to-be-marketed pharmaceutical formulation, unless tolerability or safety
concerns preclude administration of the highest dose strength.
It should be noted that the conduct of the pivotal clinical (Phase III)
studies also influences the dosing recommendations. If the efficacy studies
were performed without any special instructions regarding concomitant
food intake, this could be reflected in the text regarding “Dosage and
Administration” recommendations. However, it is highly advisable to
investigate potential food effects prior to the start of the Phase III program,

204 Sunzel
since unidentified or disregarded food effects may jeopardize a positive

outcome of the confirmatory efficacy trials.
Dose Proportionality
Dose proportionality, i.e., a proportional increase in exposure (AUC and/or
Cmax) of a drug after a corresponding increase in dose, indicates linear
pharmacokinetics of the drug. A higher exposure than predicted from the
given dose may indicate saturable metabolism or saturable first-pass effects.
A lower exposure than predicted from the given dose may indicate limitations
in the absorption processes. Early information on dose proportionality can
usually be obtained in the first safety and tolerability study. A more
confirmatory study, investigating the intended therapeutic dose range should
be performed in an adequate number of subjects and, preferably, with a
pharmaceutical formulation that is relevant to the one that will be used in
the confirmatory clinical trials in patients. Although the use of an oral solution
generates basic pharmacokinetic information regarding the drug substance,
choosing an early prototype immediate release formulation or a Phase II/III
formulation could give additional valuable information.
Choice of Dose
The dose linearity over the intended therapeutic dose range should be fully
investigated, and included in an NDA submission. However, in the early
stages of drug development the therapeutic dose range is usually not well
established, and therefore it is advisable to investigate the pharmacokinetics
of a new chemical entity over a wide, although reasonable, dose range.
Especially the upper parts of the dose range is of interest, since the break-
point for potentially clinically relevant nonlinearities in the pharmacoki-
netics of a drug should be captured and quantified as early as possible in the
development program.
An adequate number of dose levels (≥3) should be examined, but a fixed
number of dose levels are not required. It may not be necessary to repeat the
dose-linearity study with the to-be-marketed pharmaceutical formulation
unless substantial formulation changes have been made, or potential
nonlinearities have been identified. However, the reader is referred to Part B:
Biopharmaceutics for relevant information regarding waivers and
bioequivalence requirements.
Study Population
The study can be performed in healthy, adult male and female volunteers,
above 18 years of age. If the intended target population mainly consists of,

e.g., elderly patients, more valuable information may be generated by

performing the study in healthy elderly volunteers or in the target patient
population.
Study Design
A single-dose, randomized, crossover design, is the most common choice for

a dose-proportionality study. An incomplete block design, where an equal
number of subjects are randomized to receive different doses and all cohorts
together cover the full range of doses, is also an option. The latter design is
occasionally employed when the total blood volume collected from a single
volunteer would exceed standard limits of blood donations. The number of
subjects participating in the study should be based on earlier studies where
intersubject, and if available, intrasubject variabilities have been deter-
mined. Study drug should be administered with a standardized volume of
water after overnight fast, and standardized meals should not be served
until four hours post-dose.
Concomitant food intake should be avoided, unless the drug is associated
with adverse events, such as nausea or vomiting, which could be
circumvented by a small meal. It is advisable to include the rationale for
coadministration of the drug and food in the protocol. If the drug is
associated with adverse events that preclude high single doses, a titration
design where the pharmacokinetics is determined at steady state can be an
alternative.
In certain cases, a parallel-group design may be selected instead of a
crossover design, e.g., for drugs with a long terminal half-life, although a
substantially larger number of subjects may be needed compared to a
crossover design. If a crossover design has been chosen, the
investigational periods should be separated by an adequate washout
interval (>5t½) to ensure that elimination is complete before a second
dose is administered. Blood samples should be collected to adequately
describe the full plasma/serum drug concentration profile, especially the
terminal phase should be adequately described, where sampling should
be continued up to at least three to four terminal t½ of the drug and/or
active metabolites.
Data Analysis
Standard pharmacokinetic parameters (Cmax, tmax, AUQt, AUC∞, CL/F, t1/2)
are calculated by nonparametric or parametric methods for the intact drug
and major active metabolite(s). The reader is referred to the section Data
Analysis on page 199 of this chapter, for a more detailed description of the

206 Sunzel
calculations. The parameters describing exposure (Cmax and AUC) or

apparent oral clearance (CL/F) are of most interest for orally administered
drugs. For short-acting drugs, such as agents for the treatment of insomnia
or acute pain, the intial exposure (truncated AUC up to Cmax or Cmax) may be
a more relevant descriptor for dose proportionality than AUC∞.
These parameters are graphically displayed vs the administered dose,
where a straight line indicates linear pharmacokinetics over the studied
dose range. It is recommended that the analysis is performed after dose
normalization of the parameters has been performed. There is no formal
regualtory recommendations regarding the method of choice. The
interested reader can find points to consider regarding the statistical
analysis to determine dose proportionality in an article by Gough et al.
[22], where a comparison of the performace of different statistical
methods was investigated. The data should also be analyzed regarding the
similarity of the other pharmacokinetic parameters at the different dose
levels, a shift in terminal half-life or t max between doses may need
additional attention, and the potential clinical relevance of any
dissimilarities in these parameters between different doses should be
considered.
REPEATED-DOSE STUDIES
The majority of drugs are intended for chronic or multiple dose therapy in
the treatment of a specific medical condition. Even if the pharmacokinetics
has been shown to be linear over the intended therapeutic dosing interval
after single doses, this may not hold true after repeated dosing. Therefore,
the pharmacokinetics of the drug after repeated administration needs to be
investigated. Time-dependencies in the pharmacokinetics, such as
autoinduction or inhibition of the drug’s own metabolism, may occur. A
qualitative indicator can be obtained from in vitro studies or preclinical
pharmacokinetic studies in animals; however, the magnitude of the
potential time-dependency, or lack thereof, can only be assessed in vivo in
humans.
Choice of Dose and Dosage Regimen

The pharmacokinetics after repeated administration of the highest dose
level in the anticipated therapeutic dose range should be adequately
described, since more prominent changes are expected to occur at higher
dose levels. It is prudent to include one or two lower dose levels, to fully
establish the pharmacokinetic properties of the drug at steady state, after
repeated dosing. The pharmaceutical formulation of an oral dosage form

should preferably be similar to the formulation used in the later clinical

trials in the patient population. However, if the results from the single-
dose trials call for the development of a modified release or extended
release formulation, a smaller trial at an adequate dose level using an
immediate release formulation could be considered. If apparent
nonlinearities in the steadystate pharmacokinetics of the drug are
observed at a later stage, such a pilot study could be used to the
differentiate between apparent nonlinearities due to time-dependencies in
drug metabolism, and the effects of the altered release profile by the
pharmaceutical formulation.
The dosing regimen, i.e., the time-interval between doses, is governed by
the exposure (pharmacokinetic)-response (pharmacodynamic) relation-ship
of the drug. If relationship is known, the clearance and terminal t½ of the
drug can be used in the calculations of the optimal-dose regimen [1, 23].
Although the exposure-response relationship may be less well-character-
ized, the information about the pharmacokinetic properties of the drug will
aid the choice of dosage regimen. A drug with a short terminal t½ and high
clearance, where the desired effect is more likely to be related to the AUC
rather than Cmax, will require more frequent dose intake than a drug with a
longer terminal t½ and a lower clearance. The reader is also referred to
relevant chapters in the Biopharmaceutics section of this book, for pertinent
information regarding waivers and bioequivalence studies that may be
needed to fulfill all requirements for an NDA, if major changes in the
pharmaceutical formulations have been made during the development
program.
Study Population
As described elsewhere in this chapter, the study population of choice is
usually healthy, adult male and female volunteers, above 18 years of age.
The pharmacokinetics of the drug after repeated dosing should also be
studied in the intended target patient population, and compared to that of
the healthy volunteers. However, the comparison of the steady-state
pharmacokinetics of the drug between healthy volunteers and patients can
be made across studies, and a direct comparison in the same study is not
necessary.
Study Design
An open-label, randomized crossover design is usually chosen if more than
one dose-level is included in the study. The number of subjects participating
in the study should be based on data regarding variability from earlier
studies. Study drug should be administered according to the chosen dosage

208 Sunzel
regimen, based on the currently available pharmacokinetic and

pharmacodynamic information. Concomitant food and drug intake during
the investigational days is usually restricted, and the drug is administered in
the fasting state or the drug and food intake is separated by a time-interval
of approximately two to four hours.
Blood samples for drug analysis should be collected to adequately
describe the attainment of steady state (samples collected immediately
prior to the next dose intake during 3–4 dosing intervals, i.e., trough
concentrations) and the full plasma/serum drug concentration profile
during one, usually the last, dosing interval at steady state. It is
recommended that the blood sampling is continued to adequately describe
the terminal phase after the last dose intake, e.g., the collection be
continued up to at least four terminal t½ of the drug and/or active
metabolites.
Investigational periods are usually separated by an adequate washout
interval (>5t½) to ensure that elimination is complete before a second-
dose regimen is initiated. Alternate designs, where the subsequent study
periods are immediately initiated, without a washout period, should be
carefully considered, and only be used if the lack of time-dependent
changes in the pharmacokinetics of the drug has been established. An
alternate approach is to combine a single-dose and the repeated-dosing
regimen in the same subject. In that case, adequate blood sampling should
be performed after the first dose, and the repeated dosing is started
immediately after the last blood sample of the single-dose period, and
blood sampling is performed when steady state has been attained, as
described above.
Data Analysis
As described elsewhere, the standard pharmacokinetic parameters (Cmax,
tmax, CL/F, t½; in case of the administration of a single dose: AUCt, AUC∞)
are usually calculated by nonparametric or parametric methods for the drug
and major active metabolite(s). The parameters that are specific for repeated
dose administration are the AUC during one dosing interval (AUCt) at
steady state and the accumulation ratio. The latter can be directly calculated
if single-dose data also is available. The reader is referred to the section
“Data Analysis” on page 199 of this chapter, for a more detailed description
of the calculations.
The choice of analysis of the attainment of steady state, from the trough
plasma concentrations of the drug, should be stated in the protocol. If more
than one dose level is investigated, an analysis of dose proportionality
should be performed. It is advisable to include more than one dose, since an
unexpected observation of a time-dependency in a parameter, e.g., a larger

than expected AUC, may not only be caused by metabolic inhibition, but
could also be due to pharmacokinetic model misspecification. For example,
the terminal t½ may not have been correctly determined, potentially due to
lack of sensitivity in the analytical method, or the full degree of drug
accumulation in tissue has not been previously achieved after single-dose
administration. In addition to the analysis of the attainment of steady state
(and dose proportionality if applicable), the report should contain clear
graphs and tables of both individual and average data, as well as summary
statistics.
SUMMARY
In conclusion, a relatively limited number of studies are required to

adequately describe the basic pharmacokinetic properties of a drug.
Although healthy adult volunteers are usually the population of choice
for the basic pharmacokinetic studies of a drug, the validity of the data
in comparison with the pharmacokinetics of the drug in the target
patient population should also be established. A cross-study comparison
with regard to the standard pharmacokinetic parameters (Cmax, t max,
AUC, and t½) for one or two dose levels would suffice if the
pharmacokinetics are similar in the two populations. The information
that has been gathered in the studies described in this chapter is usually
included in an NDA submission. In addition to these studies,
pharmacokinetic studies in special populations or disease states, drug-
drug interactions, and bioequivalence studies, as described elsewhere in
this book, are usually included in an NDA submission. Table 1
summarizes the information that is generally expected in an NDA
submission.
It can be concluded that the choice of a study design based on careful
evaluation of previously gathered data from preclinical and/or prior
pharmacokinetic studies is essential to optimize, or minimize, the number
of pharmacokinetic studies needed in a development program. Although
the timing of the pharmacokinetic studies has not been discussed, the
pharmacokinetic information should be used throughout the development
program, since a simple description of the pharmacokinetics of a drug
serves no purpose in itself. The pharmacokinetic properties should be a
valuable instrument in the rational development of the drug. Therefore, it
is also vital to include exposure-response analyses of relevant
pharmacodynamic parameters throughout the development program, to
achieve the best possible knowledge base relevant to the therapeutic use of
the drug.

210 Sunzel
TABLE 1 Summary of the Descriptive, Basic Pharmacokinetic Information that is

Generally Expected in an NDA Submission for a New Chemical Entity
REFERENCES
1. Rowland, M.; Tozer, T.N. Clinical pharmacokinetics: concepts and applications,

3rd Ed.; Williams & Wilkins (Lea & Febiger), Media: PA, USA, 1995.
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Pharmaceuticals.” Tripartite harmonized ICH guideline (Multidisciplinary).

3. Bischoff, K.B.; Dedrick, R.I.; Zaharko, D.Z.; Longstreth, J.A. Metotrexate

Pharmacokinetics. J. Pharm. Sci. 1971, 60, 1128–1133.
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5. Mordenti, J. Man versus Beast: Pharmacokinetic Scaling in Mammals. 1986, J.
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6. Lavè, T.; Coassolo, P.; Reigner, B. Prediction of Hepatic Metabolic Clearance
based on Interspecies Allometric Scaling Techniques and in vitro-in vivo
Correlations. Clin. Pharmacokinet. 1999, 36, 211–231.
7. FDA Guidance for Industry and Reviewers (Pharmacology/Toxicology):
“Estimating the Safe Starting dose in Clinical Trials for Therapeutics in Adult
Healthy Volunteers.” DRAFT December 2002.
8. Reigner, B.G.; Williams, P.E.O.; Patel, I.H.; Steimer, J.-L.; Peck, C; van
Brummelen, P. An Evaluation of the Integration of Pharmacokinetic and
Pharmacodynamic Principles in Clinical Drug Development. Experience within
Hoffman La Roche. Clin. Pharmacokinet. 1997, 33, 142–152.
9. FDA Guidance (Clinical/Medical), posted March, 1998: “The study and
evaluation of gender differences in the clinical evaluation of drugs” (First
published as “Guideline for the study and evaluation of gender differences in
the clinical evaluation of drugs” Federal Register, Notice. 58:39406–39416,
1993).
10. Bennett, J.C. Inclusion of Women in Clinical Trials—Policies for Population
Subgroups. N. Engl. J. Med. 1993, 329, 288–292.
11. Mercatz, R.B.; Temple, R.; Sobel, S.; Feiden, K.; Kessler, D.A. Women in Clinical
Trials of New Drugs. A Change in Food and Drug Administration Policy. The
Working Group on Women in Clinical Trials. N. Engl. J. Med. 1993, 329, 292–
296.
12. Dain, J.G.; Collins, J.M.; Robinson, W.T. A Regulatory and Industrial
Perspective of the use of Carbon-14 and Tritium Isotopes in Human ADME
Studies. Pharm. Res. 1994, 11, 925–928.
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Mass Spectrom. 1999, 13, 285–293.
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17. CPMP/EWP/QWP/1401/98: “Note for guidance on the investigation of
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18. Cutler, D. Assessment of Rate and Extent of Drug Absorption. Pharmac. Ther.
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Publishing Company Inc, Lancaster, PA, USA, 1993.

10
Surrogate Markers in Drug Development
Jürgen Venitz
Virginia Commonwealth University
Richmond, Virginia, U.S.A.
INTRODUCTION
PK/PD Relationship
Several conferences and publications starting in the early 1990s until
recently have emphasized the crucial role that pharmacokinetic-
pharmacodynamic (PK/PD) modeling and the use of surrogate marker can
have in streamlining the drug development process [1–9]. In particular, the
advent of pharmacogenomics and biotechnology-derived drug products are
thought to accelerate and facilitate the use of these techniques in making the
drug development process and regulatory decision-making more rational
and efficient [5, 8].
PK/PD modeling attempts to establish quantitative (e.g., mathematical
and/or statistical) relationships between dosing regimen and pharmacologi-
cal (PD) responses, and possibly clinical outcomes (see also Chapter 11).
As shown on Fig. 1, PK relates the dosing regimens of the drug product
(e.g., dose, dosing interval, rate, and route of administration) with drug or
metabolite concentrations in the body, typically measured in plasma. Both
213
214 Venitz
FIGURE 1 Surrogate markers in clinical pharmacology (exposure-response

paradigm) and sources of variability.
dosing regimens and/or systemic concentrations are reflective of drug

exposure to the patient: The assigned dosing regimen to a patient may
reflect nominal exposure, while systemic concentrations (e.g., AUC, Cnax?
etc.) reflect systemic exposure. The latter exposure measure is more closely
related to drug/metabolite concentrations at the receptor site(s) responsible
for the drug-induced pharmacological effect(s). It also allows to compare
patients based on variability in medication adherence (compliance), as well
as drug absorption and disposition that may be affected by patient
covariates and contribute to the overall variability in drug response (see
Chapters 8 and 9).
On the other hand, PD relates the drug concentrations in the body to any
observable (multivariate) pharmacological response. A pharmacological
response can be any physiological, biochemical, or pharmacogenomic
endpoint that can be measured and is temporally and causally related to the
drug. This PK/PD relationship is also referred to as the exposure-response
(ER) relationship. Any variability in this relationship within and between
patients contributes to the overall variability in drug response. In general,
the PD responses are mediated by the mechanism(s) of action (MOA) of the
drug. Nevertheless, a drug may have additional PD effects that are not
mediated by the primary MOA such as hepatotoxicity.
Finally, the PD response(s) may be related to the ultimate clinical
outcome(s), i.e., clinical efficacy and toxicity. If so, these (special) PD
responses are surrogate markers that may substitute for clinical outcomes,
since they usually are easier to measure and allow appropriate
dosingregimen adjustments without having to accept adverse clinical
outcomes.

Surrogate Markers in Drug Development 215
It is one of the basic tenets of clinical pharmacology that an exposure-

response relationship exists for clinical outcomes; namely, that changing the
dose, etc., (exposure) has a tangible impact on outcomes. As a corollary, it is
essential to optimize the dosing regimen according to the known PK/PD
covariates.
Surrogate Markers
The choice of the term “marker” used to indicate a marker of biological
drug response (biomarker) or the clinical outcomes (surrogate marker)
originates from clinical medicine, where markers are used to indicate
absence or presence of a disease (diagnostic purpose) and/or predict the rate
and extent of disease progression (prognostic purpose).
As shown in Fig. 2, these markers are strongly tied to our understanding
of the pathophysiology of the disease (POD) being treated. The use of
markers in clinical medicine for diagnostic or prognostic purposes is
justified based on epidemiological and/or interventional clinical studies that
assess their ability to predict clinical outcomes.
Dosing-Regimen Optimization
Using the PK/PD framework discussed above along with the use of
surrogate markers allows the optimization of dosing regimen in the drug
development and in clinical practice.
Figure 3 illustrates typical exposure-response relationships for clinical
efficacy and toxicity. Depicted is the percentage of patient responding (i.e.,
showing either efficacy or toxicity) as function of an exposure measure. In
the simple case, these relationships can be thought of as dose-response
curves for efficacy and toxicity. Both exposure-response curves show a
sigmoidal relationship due to the above mentioned population variability in
PK and PD. An optimal exposure (e.g., dose) is designed to minimize the
likelihood of toxicity while maximizing the likelihood of clinical efficacy.
FIGURE 2 Surrogate markers in clinical medicine (epidemiology).

216 Venitz
FIGURE 3 Example of exposure-response relationships for clinical efficacy and

toxicity.
Knowledge of this ER relationship allows the rational selection of an

optimal exposure. Note that similar relationships are expected to exist for
biomarkers and surrogate markers as well, but their shape and variability
may be quite different.
Therefore, it is essential during the early clinical drug development
process to identify important clinical covariates (such as age, gender, renal
function, comedications, etc.) of the PK and PD drug properties, along with
a potential surrogate marker of efficacy and/or toxicity. The former
information allows the rational selection of a patient-specific dosing
regimen (dose individualization) while the latter allows continuous
assessment of the therapeutic regimen and can trigger dosing regimen
adjustments intended to avoid toxicity and/or improve efficacy.
In clinical practice, using the above information provided on the
approved drug-product label permits the prescriber to individualize the
patient-treatment regimen and to continuously monitor the treatment
success using the surrogate marker (therapeutic drug monitoring, TDM).
This is particularly important for diseases and drugs where the ultimate
clinical outcome is mortality, and a suboptimal dosing regimen is likely to
result in excess mortality due to either lack of efficacy or toxicity. Table 1 is
an incomplete list of some biomarkers/surrogate markers used in drug
development and clinical practice (see also Chapter 12).

TABLE 1 Examples of Bio-/Surrogate Markers and their Basis in POD and/or MOA
(see text for abbreviations)
The QTc-interval (measured on the electrocardiogram) has been shown

to predict the occurrence of fatal arrhythmias (torsades de pointes, TdP)
associated with quite a few drugs, some of which have recently been
withdrawn from the marketplace due to insufficient risk/benefit ratio
(namely, terfenadine, astimazole, etc.). Prolongation of the QTc-interval is
thought to be a precursor of TdP.
Plasma cholesterol (a biochemical measure) and blood pressure (a
physiological measure) are some of the oldest surrogate markers. Initially,
during epidemiological studies in the 1960s (Framingham), elevated levels
of these markers were shown to be associated with increased cardiovascular
mortality and morbidity. Later on, in prospective interventional clinical
studies using blood pressure or cholesterol-lowering medications and diets,
the markers were shown to be causally related to cardiovascular outcomes.
Additionally, mechanistic studies elucidated the POD, i.e., the
pathophysiological chain of events leading from hypercholesterolemia and
hypertension to cardiovascular morbidity and mortality.
Pulmonary function tests such as FEV1 and PEF are used in clinical
practice to assess the progression of chronic bronchial asthma as well as to
monitor treatment with steroids and bronchodilators, to change drug and/
or dose, if necessary.
The CD4-lymphocyte count in peripheral blood was the first surrogate
marker used in the marketing approval of AZT (zidovudin) for the

218 Venitz
treatment of HIV infection in the late 1980s. At that time, higher CD4
counts were found to be negatively correlated with disease mortality.
Mechanistic studies had shown the role of CD 4 lymphocytes in the
pathophysiology of HIV infection. Due to the poor prognosis of the disease
and the lack of any effective treatment, AZT was approved based on
clinically significant increases in CD4 counts rather than a proven mortality
benefit, which was shown later in phase IV studies. Currently, the HIV viral
load in plasma is the accepted surrogate marker of disease progression and
treatment success, both in drug development and clinical practice. In the
future, HIV pheno-/genotyping may be an even better predictor of clinical
outcomes.
Cyclosporine (CsA), used to prevent organ rejection, is known to have a
high level of between- and within-patient PK variability, and the
consequences (clinical outcomes) of inappropriate exposures are severe,
namely organ rejection (lack of efficacy) or renal toxicity. As a result, CsA
serum concentrations are measured (as a surrogate endpoint) and used to
adjust the dosing regimen, if necessary.
The International Normalized Ratio (INR), an in vitro coagulation test is
used successfully in the TDM of warfarin therapy, an oral anticoagulant.
Warfarin is known to be associated with high PK and PD variability
between and within patients; the consequences of inadvertently low or high
exposure of warfarin can be disastrous, namely ischemic or hemorrhagic
stroke. INR values have been shown to predict these clinical outcomes, and
target INR ranges have been established to guide warfarin dosing. It is
noteworthy to recognize that the INR predicts both efficacy and toxicity
since both outcomes are due to the MOA of warfarin.
Finally, blood hematocrit is used as a surrogate marker in the treatment
with erythropoietin (epo) since it does predict quality of life, and epo is a
very expensive treatment mandating appropriate dose selection and
adjustments in clinical practice.
DEFINITIONS
Consensus has been reached on the terminology of the different markers

[6, 7, 9]:
Terminology of Markers
1. Biomarker (Intermediate Endpoint): A biological (pathophysiological or
pharmacological) indicator that can be measured as a result of a therapeutic
intervention. It may or may not be related to clinical outcomes, but is
involved in the chain of events in the POD and/or MOA the drug.

2. Clinical Outcome: A clinically accepted indicator of disease state/

progression, e.g., survival, morbidity, symptom scores, etc. Clinical
outcomes are measures of the efficacy or safety/toxicity of a drug.
3. Surrogate Endpoint/Marker: A biomarker that predicts clinical
outcomes as accepted by the scientific, medical and regulatory community.
It may substitute for clinical outcomes in the drug development process
(dosing-regimen and dosage-form optimization and possibly drug approval)
and in clinical medicine (TDM). At least some of the variability in clinical
outcomes is explained by changes in surrogate markers. [6, 7]
A biomarker (candidate/putative surrogate endpoint in the drug
discovery/development process) can achieve surrogate endpoint status if
properly evaluated. Evidence to support that linkage integrates information
from multiple sources such as molecular biology, pathophysiology of the
disease, mechanism of action of the drug candidate, clinical trials, and
epidemiological studies.
EXPOSURE-RESPONSE RELATIONSHIP
The exposure-response relationship measures the association between

responses (clinical outcomes, surrogate markers, biomarkers) and drug
exposure (dose, systemic concentrations, etc.). This relationship can be
modified by clinical covariates, both intrinsic and extrinsic. Clinical
pharmacology studies help in elaborating the shape and variability of this
relationship (see Chapter 11).
Characteristics of Markers
Based on the measurement scale that they are measured on, PD markers can
be classified as follows:
1. Graded Response: A quantifiable PD marker (such as an in vivo
physiological response or in vitro test) that is causally and temporally linked
to drug treatment and related to drug exposure (ER relationship), e.g.,
blood pressure, serum cholesterol, INR, etc. These endpoints are usually
chosen based on the MOA of the drug and known receptor-mediated
physiological or biochemical responses.
A graded response is a continuously scaled variable, can be measured
repeatedly within the same individual, and is typically used for PK/PD
modeling, particularly preclinically and in phase I/II.
2. Challenge Response: A quantifiable, graded response to a standardized
exogenous challenge agent that is modified by administration of the drug of
interest and related to drug exposure, e.g., exercise-induced tachycardia (to
assess ß1-blocker activity), and histamine-induced broncho-constriction (to

220 Venitz
assess H1-blocker activity). These markers are based on the MOA of the
drug and sometimes on the POD. This kind of markers usually requires
additional special clinical testing and are rarely used in clinical practice for
dose adjustment.
A challenge response is a continuous variable (e.g., percent inhibition
relative to baseline or placebo). It requires additional interventions, may not
be repeated often within the same individual during a dosing interval, and
contributes possibly unacceptable additional safety issues in phase I/II
studies. However, it can be used for PK/PD modeling.
3. Categorical Response: A “Yes-or-No” response due to drug
administration that can be related to drug exposure, e.g., death, organ
rejection, incidence of AE. This type of response is usually a clinically
relevant outcome based on the disease progression in question, regardless of
the MOA. It can be measured as part of clinical practice, but does not allow
treatment adjustment.
However, it can be measured only once within a given patient. It is a
nominal variable that is not very informative statistically and requires a
large sample size. It is used in phase II/III studies along with population PK/
PD analysis.
4. Time-to-event Response: Time-to-event that is related to drug
exposure, e.g., survival time, time to relapse. This type of response is usually
a clinically relevant outcome based on the disease progression in question,
regardless of the MOA. It can be measured as part of clinical practice, but
does not allow treatment adjustment.
It is a censored continuous variable that can be measured only once
within a patient, is not very informative, and requires a large sample size in
phase II/III studies along with population PK/PD analysis.
5. Event Frequency/Rate Response: Frequency of clinical events related
to drug exposure, e.g., seizure frequency, frequency of cardiac
arrhythmias.
It is a censored continuous variable that can be measured more than once
within a patient; however, is not very informative, and requires a large
sample size in phase II/III studies and population PK/PD analysis.
USE AND BENEFITS IN DRUG DEVELOPMENT
Markers in Drug Discovery and Development

Biomarkers have to be identified early during the drug-discovery process
and evaluated/validated systematically throughout the subsequent drug-
development process:

1. Discovery: Potential biomarkers/surrogate makers should be selected

based on the current mechanistic understanding of the pathophysiology of
the disease and proposed mechanism of action of the drug candidate based
on theoretical considerations and/or experimental evidence. Additional
thought should be given to potential toxicity markers not associated with
the known MO A (e.g., other drugs in the same pharmacological class,
known toxicities in disease, known biomarkers in the disease).
2. Preclinical Development: The in vitro binding of the drug candidate
to receptor/enzyme and/or in vivo or ex vivo functional testing (enzyme
activity or receptor intrinsic activity) should be evaluated for feasibility
as markers across various species, including humans. Ex vivo or in vivo
challenge paradigms based on the MOA should be considered. As part of
the preclinical workup, ER relationships for potential markers of
efficacy/toxicity should be established. This will allow interspecies
scaling and optimal selection of starting dose and dose-escalation
increment or even a PD-guided study design for phase I for the first time
in human studies.
3. Clinical Development: In phase I, in vivo testing/challenge paradigms
in healthy volunteers should be considered to establish the ER relationship
in low-population-variability setting. In phase II, demonstration of
changes of biomarkers in the expected direction may serve as proof-of-
concept (POC) suggesting clinical efficacy of the drug candidate, and help
in making important Go-No Go decisions. Throughout the phase II stage,
biomarkers should be correlated with short-term clinical outcomes in the
target patient population; attempts should be made to establish ER
relationships for biomarkers/short-term clinical outcomes. This
correlation between biomarker and accepted (approvable) clinical
outcomes should be quantitated in the phase III program, and important
clinical covariates affecting outcome and marker should be identified. If
necessary, the surrogate marker can be used for therapeutic monitoring of
postmarketing in clinical practice. Demonstrated ER relationships with
biomarkers or surrogate markers will also be useful in phase IV to assess
new dosing regimens, dosage forms, and special populations (namely
pediatrics).
Benefit of Using Markers in the Drug Development Process

1. Identification of Biological Sources of Variability in Drug Response: For
rational drug development, it is important to understand the contribution of
PK or PD variability to the overall population variability in drug response
(for dose individualization and TDM).
2. Physiological Interpretation of PK/PD Parameters: Appropriate
physiological interpretation of PK and PK/PD parameters early in the drug

222 Venitz
development (in-vitro, preclinical, phase I) allows appropriate interspecies

scaling and informative “Go-No Go” decisions [8].
3. Identification of Relevant PK/PD Covariates: The PK/PD approach
throughout drug development assists in anticipating and identifying
important patient factors, e.g., age, gender, concurrent diseases, and
comedications, that may require dose individualization/therapeutic
monitoring in the target population (see Chapters 8 and 9).
4. Rational Optimization of Dosage Forms and Dosage Regimens:
Understanding of the intrinsic PK/PD characteristics with an acceptable
biomarker and sources of population variability permits better design of
dose-finding studies in phase I and II as well as rational development of
appropriate dosage forms. It may also be useful in selecting optimal backup
compounds to the lead compound.
5. Rational Labeling Decisions: Appropriate PK/PD modeling with an
acceptable biomarker helps assessing and interpreting the PK results of
“equivalence” studies, i.e., food-effect, chronic renal and hepatic
disease-effect, and drug-drug interaction studies, by allowing to define a
target range of “no clinically significant PK difference” (“What-If”
Scenarios).
It is the PK/PD information gained in the drug development process that
drives the final clinical dosing-regimen recommendations (particularly dose
individualization and therapeutic monitoring) in the product label.
6. Marketing Approval: PK/PD studies with an acceptable surrogate
marker may provide supportive (“confirmatory”) evidence for drug
approval in lieu of an (second) adequate and well-controlled phase III
clinical trial, particularly for extension into special populations (e.g.,
pediatrics) or new dosage forms. However, this is likely to occur only if
the surrogate marker has been accepted after comprehensive evaluation
and other drugs in the same class have shown benefits in clinical
outcomes.
Assessment of Measurement Performance of Biomarkers

In addition to their validity, the measurement techniques for biomarkers
and surrogate markers have to be assessed for their reliability in practice
[6, 10]:
1. Sensitivity: The ability of the of the measurement technique to detect
small changes in the marker.
2. Specificity: The ability of the measurement technique to differentiate
drug-induced changes from spontaneous changes in the marker.
3. Reproducibility (Accuracy and Precision): The ability of the
measurement technique to provide consistent results throughout clinical
studies and development programs.

Tests used in clinical practice may not necessarily be rigorous and rugged
enough to measure biomarkers and surrogate markers as part of a drug
development. Additional technology may be needed to improve the
reliability of the measurement techniques.
Integration of Knowledge Gained during Development Process

PK/PD modeling with biomarkers and/or surrogate markers use and
combine quantitative information from various disciplines such as
pharmacology, toxicology, pathophysiology, clinical pharmacology, and
biopharmaceutics. This allows each discipline to provide important
input in each phase of the drug development. Throughout the
development, information will need to updated, PK/PD models revised,
PK/PD model parameters adjusted, and biomarkers evaluated for their
further use.
If done consistently, the PK/PD database can serve as the foundation of
clinical trials simulations (see Chapter 11). Clinical trials simulation uses
PK/PD models and model parameters (and their statistical distributions) to
predict clinical outcomes as function of dosing regimens or study designs.
This is extremely useful in optimizing clinical study designs and sample size
for phase II/III studies.
LIMITATIONS
Limitations of PK/PD Modeling using Surrogate Markers

1. Validation/Evaluation of Surrogate Endpoints: What is the relationship
between changes in (surrogate) PK/PD endpoints and clinical acceptable
efficacy and/or safety outcomes? The validity of PK/PD modeling depends
on both surrogate endpoint validation and PK/PD model validation.
Surrogate endpoint validation is a continuous process that should start at
the preclinical stage; it requires front-loading of the drug-development
process.
2. Incorporation of Long-term Disease Progression and
Subpopulations: If the PD endpoint is clinically meaningful (surrogate
marker), the effect of disease progression in patients with the disease may
have to be incorporated as baseline PD model in the PK/PD model. If
possible, the endpoint should be demonstrated to be meaningful across
subpopulations of patients.
3. Long-term Changes in PK or PK/PD Relationship (Time-invariance):
Typically, the PK model and the population parameter estimates are
obtained from single-dose or short repeated-dose studies, which do not

224 Venitz
reflect the reality of chronic treatment of most chronic diseases. However,

the PK may change over time, e.g., due to autoinduction or other secondary
drug-induced changes in PK.
Typically, the intrinsic ER relationship (e.g., effect-biophase concen-
tration relationship) is assumed stationary, i.e., invariant with time [10].
This means that at (PK and PD) steady state, there is a constant relationship
between effect and plasma concentration. However, there is an increasing
number of drugs where this is not necessarily true, and PD tolerance or
resistance develops as function of time and dosing regimen, and the
“intrinsic” PK/PD relationship changes with time.
4. Empirical vs. Mechanistic PK/PD Modeling: The objective of the PK/
PD modeling exercise determines the use and validation of PK/PD models:
Empirical models may be validated for their predictive ability, but do not
allow interpretation of their model parameters (if parametric), i.e., the
system is considered a “black box”. On the other hand, mechanistic models
allow estimation of meaningful PK/PD parameters, but the data obtained
from typical clinical studies may prevent accurate and precise parameter
estimation.
5. PK/PD Model Validation: PK/PD model validation is a clinical
pharmacology issue based on statistical concepts. However, internal model
validation is only a part of PK/PD model validation: The surrogate PD
endpoint used has to be clinically validated (external validation), i.e., has to
be linked to clinically acceptable efficacy or safety outcomes (accepted/
approved by the medical specialists). There is growing research activity
attempting to link surrogate PD endpoints (typically continuously scaled
variables) mathematically to clinically relevant outcomes (typically catego-
rical variables), as shown in clinical trials simulations (e.g., QT c -
prolongation and likelihood of TdP).
Any PK/PD model, be it empiric or mechanistic, parametric or
nonparametric, can and has to be validated for its intended use: Validation
means assessment of descriptive performance (interpolation), predictive
performance (extrapolation), and estimation of meaningful PK and PK/PD
parameters that can be interpreted. In general, the PK/PD models have to be
predictive (within certain constraints of dosing regimens and time) to be
useful, but not necessarily mechanistically interpret able.
Potential Pitfalls of Surrogate Markers

Since surrogate markers are expected to substitute for clinical outcomes, the
following situations may occur:
1. Perfect Surrogate Endpoint: The full effect of the (drug)
intervention on clinical outcome(s) is reflected and predicted by
corresponding changes in the marker (perfect correlation). This ideal

scenario does not exist (yet), and probably never will, since no single
marker can reflect the entire (multivariate) pathophysiology of a disease
or pharmacology of a drug [8].
2. Acceptable Surrogate Endpoint: Changes in the marker reflect only
partially the (drug) intervention effect on clinical outcomes, e.g., cholesterol
for statin drugs, blood pressure for antihypertensives, HbA 1c for
antidiabetics, etc. These are endpoints that, based on available evidence, are
accepted by the scientific and medical community to substitute for clinical
outcomes, both in the drug development and in clinical practice.
3. False Positive Endpoint: The drug intervention affects the marker
favorably, but has an unfavorable effect on clinical outcome, e.g.,
premature ventricular contraction (PVC) frequency for antiarrhythmic
agents: The placebo-controlled, randomized, double-blind Cardiac
Arrhythmia Suppression trial (CAST) demonstrated that various
antiarrhythmic agents did suppress PVC frequency in patients with
cardiac arrhythmia, which had been thought to predict improved clinical
outcome, namely mortality. However, CAST showed excess mortality in
the active-treatment groups relative to the placebo group (most likely due
to the arrhythmogenic effects of the drugs), disproving PVC suppression
as a surrogate marker.
From a regulatory point of view, this appears to be the major concern in
using surrogate endpoints to approve drug products for marketing, and
necessitates the requirement of adequate and well-controlled clinical phase
III trials to demonstrate efficacy.
4. False Negative Endpoint: The drug intervention affects the marker
unfavorably (or not at all) but has a favorable effect on clinical outcomes,
e.g., Prostate-specific antigen (PSA) in treatment of prostate cancer.
Evaluation/Validation of Surrogate Endpoints

Evaluation or validation of biomarkers to serve as candidate surrogate
markers is an ongoing process starting in the drug-discovery stage and
continuing throughout the drug-development process. The extent of
validation depends on the intended use of the marker; e.g., if the surrogate
marker is intended to be used for drug approval (in lieu of clinical evidence
of efficacy or toxicity), there is a high burden of evidence to that effect. On
the other hand, if the biomarker is used for internal decision-making, such
as Go-No Go after POC or other phase I/II studies or dose selection for
phase II/III studies, less evidence to support their use is necessary. Evidence
to support the contention that a biomarker may be a surrogate for clinical
outcomes can be derived from the following studies:
1. Mechanistic studies identify the biomarker(s) based on our knowledge
of the pathophysiology of the disease and the mechanism of action of the

226 Venitz
drug for its efficacy. However, in general, our incomplete understanding of

POD and MOA makes this level of evidence the weakest. Furthermore,
clinical toxicities may have different (unknown) MOAs unrelated to the
MOA involved in clinical efficacy.
2. Epidemiological studies demonstrate a correlation (not causation)
between biomarker and clinical outcome. These studies are typically
designed to stratify patients based on their risk of disease progression and
demonstrate the diagnostic and/or prognostic use of markers, typically
based on our understanding of the POD.
3. Clinical pharmacology studies establish a (temporal and causal)
relationship between biomarker and drug administration (ER relationship).
This is strong evidence that the drug treatment (rather than other extrinsic
covariates) is responsible for the biomarker changes. In conjunction with 1
and 2, clinical pharmacology studies strengthen the validity of a biomarker
as a surrogate marker.
4. Clinical intervention trials (with the gold standard of a prospective
randomized clinical trial) demonstrate a (causal) link between changes in the
biomarker and clinical outcomes. This helps establish at least the (partial)
predictability of clinical outcomes from the biomarker and allows the
biomarker to achieve surrogate endpoint status.
CONCLUSIONS
The impact of PK/PD modeling on the clinical development process and its
acceptance by the scientific and regulatory community depends on the
acceptance of appropriate surrogate endpoints and the validity of the
modeling practice.
Due to our incomplete understanding of pathophysiology of most
diseases and mechanism of action for efficacy of drugs, the use of surrogate
endpoints may be limited, particularly as markers of toxicity (e.g.,
hepatotoxicity).
Evaluation of candidate surrogate endpoints has to start early in drug
discovery and continue throughout the preclinical and clinical development;
it requires additional resources and commitment to interdisciplinary
collaboration.
The potential payoff of PK/PD modeling using surrogate endpoints lies in
the streamlining of the clinical development and regulatory approval
process, and improved therapeutic labeling and monitoring in clinical
practice. The approach may also provide supportive evidence of efficacy
and/or safety to allow marketing approval under special circumstances (e.g.,
dosage form changes, pediatric population etc.).

REFERENCES
1. Peck, C.C.; Barr, W.H.; Benet, L.Z.; Collins, J.; Desjardins, R.E.; Furst, D. E.;
Harter, J.G.; Levy, G.; Ludden, T.; Rodman, J.H.; Sanathanan, L.; Schentag, J.J.;
Shah, V.P.; Sheiner, L.B.; Skelly, J.P.; Stanski, D.R.; Temple, R.J.; Viswanathan,
C.T.; Weissinger, J.; Yacobi, A. Opportunities for Integration of
Pharmacokinetics, Pharmacodynamics and Toxicokinetics in Rational Drug
Development. Clin. Pharmacol. Ther. 1992, 51 (4), 465–473.
2. Reigner, B.G.; Williams, P.E.O.; Patel, I.H.; Steimer, J.L.; Peck, C.; van
Brummelen, P. An Evaluation of the Integration of Pharmacokinetic and
Pharmacodynamic Principles in Drug Development. Clin. Pharmacokinet.
1997, 33 (2), 142–152.
3. Derendorf, H.; Lesko, L.; Chaikin, P.; Colburn, W.; Lee, P.; Miller, R.; Powell,
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Modeling in Drug Research and Development. J. Clin. Pharmacol. 2000, 40, 1–
19.
Drug Development: Opportunities for Better Candidate Selection and
Accelerated Evaluation in Humans. Pharm. Res. 2000, 17 (11), 1335–1344.
5. Galluppi, G.R.; Rogge, M.C.; Roskos, L.K.; Lesko, L.J.; Green, M.D.; Feigal,
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6. Colburn, W.A. Optimizing the Use of Biomarkers, Surrogate Endpoints and
Clinical Endpoints for More Efficient Drug Development. J. Clin. Pharmacol.
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7. Biomarkers Definitions Working Group. Biomarkers and Surrogate Endpoints:
Preferred Definitions and Conceptual Framework. Clin. Pharmacol. Ther. 2001,
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8. Lesko, L.J.; Atkinson, A.J., Jr. Use of Biomarkers and Surrogate Endpoints in
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10. Venitz, J. Pharmacokinetic-Pharmacodynamic Modeling of Reversible Drug
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Correlations, Derendorf, H., Hochhaus, G., Eds.; 1st Ed.; CRC-Press: Boca
Raton, FL, 1994; 1–34.

11
Population Pharmacokinetic and
Pharmacodynamic Analysis
Jogarao V.S.Gobburu
INTRODUCTION
One of the critical objectives of clinical pharmacology is to individualize the

dosing recommendations by estimating the population characteristics, for
instance the central tendency and the variability, of the fundamental
pharmacokinetics (PK) and pharmacodynamic (PD) parameters in the
target population. Individualization of dosage includes describing the
variability in the PK and PD parameters using covariates such as body
weight, age, gender, disease state, concomitant medication(s), etc. In
addition, the regulatory agencies and the pharmaceutical drug sponsors use
population PK/PD analyses for a variety of other purposes through the drug
development process. These include drug candidate selection, dose
selection, clinical trial design, gaining insights into clinical trial outcomes
and others.
The U.S. Food and Drug Administration (FDA) utilizes population
analyses as an aid in making regulatory decisions at almost all stages of the
investigational new drug (IND) and new drug application (NDA) review
processes. The leadership of the FDA in making the current drug
229
230 Gobburu
development process more efficient is reflected in the many guidances that

are issued for industry to date. The FDA is the first institution to set up a
pharmacometrics group exclusively for the purpose of reviewing and
conducting research in PK/PD modeling and simulation (M&S) related
topics. The aim of this chapter is to briefly present the population analyses
methods and discuss some specific applications of the same in regulatory
review processes.
DATA AND DESIGN
Clinical trial designs dictate the data collection and analysis methods. Every
clinical trial is conducted to answer a set of questions. Clinical trial
protocols explicitly state how, when, and what to measure in a given
individual in order to analyze the data in a prespecified manner. Hence, the
analysis plan is an integral part of the experimental design. There are two
broad types of data that could be collected in clinical trials—experimental
and observational. Many PK/PD measurements are typically collected from
a clinical trial that is conducted only in a small number of subjects over a
relatively short duration of time. Data from such studies are called as
“experimental data.” Studies performed to evaluate the effect of food, renal/
hepatic impairment, or gender on the pharmacokinetics of a drug (but not
part of a large trial evaluating the clinical effect of the drug) are trials where
experimental data (10 to 20 samples per individual) are collected. Data from
each of the subjects can be analyzed independent (in most cases) of the
others and summarized.
On the contrary, when the objective of the trial is to evaluate the
effectiveness and safety of a drug in a large number of patients, obtaining 10
to 20 samples per subject may be impossible. But, a few measurements can
be performed as part of the routine examination of each of the patients.
These measurements are called as observational data. It is almost impossible
to analyze the data from each patient separately. Some of the reasons
include repeated measures, imbalance, and confounding correlation
between the design and outcome [1]. Pharmacokinetic information without
adequate understanding of the pharmacodynamics of a drug is futile. The
design of the large clinical trials that probe into the pharmacological actions
of the drug, hence, needs some discussion.
Although there are several types of designs used to evaluate effectiveness
and safety of a drug, the most widely used designs include—parallel, cross-
over, and titration. In a parallel design trial, patients are randomized into
cohorts who receive one of the several treatments (control, dose 1, dose 2, or
dose 3). Such a design will offer the population, rather than the individual,

Pharmacokinetic and Pharmacodynamic Analysis 231
PK/PD characteristics. The advantage of such a design is the lack of

confounding factors such as time (carry over effects) and design dependent
outcomes.
According to a cross-over design, each patient would receive all the
possible treatments. Therefore, a cross-over design is the most powerful
design if deducing the individual concentration (or dose)-response curves is
the ultimate aim. The disadvantages of this approach are that of its longer
trial duration, possible carry-over effects from previous doses, and the need
for sophisticated data analysis (nonlinear mixed effects modeling).
The titration design ensures that the patients usually start at a relatively
low dose and the dose is increased gradually until either no additional
benefit is observed or dose-limiting toxicity occurs. This design closely
resembles the clinical practice and the individual PK/PD character-istics can
be obtained. The major disadvantage of this design is that of the possibility
of an inverted U-shaped PK/PD relationship, as an artifact. The patients
who are less sensitive to the drug need higher doses of the drug, making it
appear as if the response decreases after a certain dose. Data analysis using
conventional methods such as ANOVA fails and the use of sophisticated
modeling techniques is required.
The control group consists of either active treatment(s) or a placebo,
depending upon the type of disease. Where administering a placebo is
considered unethical (for example, AIDS trials) active treatment serves as
the control group. The trial subjects could be randomized to dose, drug
concentration, or effect elicited by the test drug. The trials are, thus, called
as randomized dose controlled (RDCT), randomized concentration
controlled (RCCT), and randomized effect controlled (RECT) trials,
respectively. The RDCTs are the most prevalent due to the relative ease of
executing a trial. The test dose(s) are randomly administered to the subjects
and data are collected throughout the trial. The so-collected data are then
analyzed using an appropriate method (see the following section). In an
RCCT [2], the subjects are randomized to a set of prespecified (usually
plasma) concentration levels. These target concentration levels are selected
based on the PK/PD relationship characterized in previous trials/
experiments. The RCCT requires a dose-titration period where the dose to
ensure that the concentrations lie within a target range (e.g., 5±0.5 µg/L) is
identified. The requirements to conduct such a trial include: (1) availability
of prior information to select the appropriate target concentration ranges, (2)
availability of an efficient and sensitive analytical assay method with a short
turnaround time, and (3) availability of enough strengths of the formulation
to allow for the necessary dose adjustments. In an RECT [3], the subjects are
randomly assigned to a set of prespecified target effect levels. Based upon the
prior knowledge about the drug’s PK/PD, sampling is conducted and the dose

232 Gobburu
is adjusted accordingly. The requirements to conduct such a trial are similar

to that of RCCT except that in an RECT the effect is targeted. Drugs whose
PK have a large unexplained variability are candidates for RCCT and drugs
whose PD have a large unexplained variability could be candidates for
RECT. When the measured effect (desired/undesired) is symptomatic (those
which are “felt” by the patients, e.g., pain, nausea, etc.), RECT could be
applicable. When the symptoms are not obvious, RCCT could be a better
choice. Unfortunately, there are fewer drug development plans that utilized
RCCT or RECT designs.
POPULATION ANALYSIS METHODS
Types of Models
First, an attempt will be made to define a few widely used terms that are
needed for the clarity of discussions. All PK (or exposure)/PD (or response)
models are made up of several components or sub-models. While “PK” need
not be defined, “PD” encompasses drug activity (both desired and undesired
effects) as measured by biomarker(s), surrogate(s), and/or clinical end
points. The PK/PD sub-models, by and large, can be classified based on their
function (descriptive and predictive) and principle (mechanistic and
empirical).
Descriptive (Sub) Model. A model or a sub-model whose representation,
essentially, confines its use to the range of dependent variable(s) used to
build the (sub-) model. Example: A linear concentration-effect relationship
may not be able to extrapolate beyond the range of concentrations studied.
Predictive (Sub) Model. A model or a sub-model whose representation
allows its use to “predict” within and beyond the range of dependent
variable(s) used to build the (sub-) model. Example: An Emax type
concentration-effect relationship can be used to extrapolate beyond the
range of concentrations studied.
Mechanistic (Sub) Model. A model or a sub-model whose structure and
parameterization allow direct and/or indirect linkage to physiological
processes. Example: An allometric equation to relate body weight and the
clearance of a drug.
Empirical (Sub) Model. A model or a sub-model whose structure and
parameterization allow no direct and/or indirect linkage to physiological
processes. Example: A linear model to relate body weight and the clearance
of a drug.
We note the overlap in the definitions to differentiate models based on
function and principle. But there may be cases when a model is empirical

(mechanistic) in principle but predictive (descriptive) in function. An example

would be that of the dual cosine function used to describe the circadian
rhythm in most biological processes. Most known models have a
combination of the different sub-models.
Basic Framework
The hierarchy in the population analyses is—population (fixed effects),

individual (random effects), and then each observation (residual error). A
complete population PK/PD model usually constitutes of four structural and
three statistical (error) models. The four structural models include: (1) PK
model, (2) disease progression model, (3) PD model, and (4) covariate (or
prognostic factor) model. The parameters of these models are called as
“fixed effects.” Examples of fixed effects include the typical value of
systemic clearance in a 70-kg person and the mean potency of the drug. The
three statistical models include: (1) inter-individual variability (IIV) model,
(2) inter-occasion variability (IOC) model, and (3) residual error model. The
parameters of the IIV/IOC model are called as “random effects.” The
random effects models assume that the inter-individual errors (η) are
distributed with a mean zero and a variance ω2. The residual error model
assumed that the measurement (and model mis-specification) errors are
distributed with a mean zero and a variance σ2. Nonlinear “mixed” effects
models deal with both fixed and random effects simultaneously, hence the
name.
The framework of the mixed effects models is illustrated in Fig. 1.
Consider a one-compartment model when the drug was given as an
intravenous bolus. Let us also assume that the volume of distribution (V) is
identical in every individual (no inter-individual variability). The concen-
tration in the “ith” subject at the “jth” time point can be described using the
following equations:
(1)
CLi—CLPoP+ηCL,i (2)
Where CLi is the estimated clearance of the “ith” subject, CLPOP is the
estimated population mean clearance, ηCL,i is the difference between the
population and individual clearances, and εij is the residual error of the “jth”
sample of the “ith” subject. The ηCL values are assumed to follow a normal
distribution with a mean zero and variance ω2CL. The εij values are assumed
to follow a normal distribution with a mean zero and variance σ2.

234 Gobburu
FIGURE 1 The basic framework of nonlinear mixed effects modeling. Consider the
“ith” observation in the “ith” subject. The difference between the observed
concentration (solid circle) and the individual predicted concentration (broken line)
is due to the fact that the “ith” individual’s clearance (CLi) is different from the
population clearance (CLPOP) by a value of ηCL,i. An additional source of variability is
the residual error (εij) which is primarily due to model mis-specification and
measurement error. The ηCL values follow a normal distribution with a mean zero
and variance ω2CL. The eij values follows a normal distribution with a mean zero and
variance σ2. According to the present example, the NM model would estimate the
parameters—CLPOR ω2CL, and σ2.
Of the several population analyses techniques, the most popular are: (1)
naïve pooled analysis, (2) two-stage analysis (TS), and (3) nonlinear mixed
effects analysis (NM). The naïve pooled analysis is performed by pooling
data from all subjects (as if all the data are from a single “giant” subject). A
minor variation of this method involves analysis of the mean data. Both the
methods provide only the central tendency of the model parameters and no
random effects are estimated. These methods are applied more routinely
when dealing with preclinical data. Naive pooled analysis is appealing
because of its simplicity. No sophisticated software is required. The fact that

the random effects cannot be estimated and inter-individual variability

cannot be accounted using covariates (such as body size, age, etc.) makes the
potential of naïve pooled data modeling very limited.
The TS method is a reasonably powerful method to estimate both the
central tendency and inter-individual variability. The first stage involves the
estimation of the individual parameters and the second stage involves the
estimation of the population mean and variance of the parameters, after
adjusting for covariates if necessary. The TS method requires that enough
number of samples (greater than the number of model parameters) per
subject are collected, as is the typical case with experimental data. This
method assumes that the individual parameters, estimated in stage one, are
the true values for the calculations in stage two. By and large, this is a
relatively minor concern. The more serious drawbacks include modeling
sparse data from observational studies and modeling concentration (or
dose)-dependent nonlinear processes. Consider a drug whose elimination
follows Michaelis-Menten type kinetics. The data from the lower doses (or
higher doses) alone may not render enough information to estimate both the
maximal velocity (Vmax) and concentration for half-maximal velocity (km).
The same argument applies when estimating the parameters of an Emax
model.
Nonlinear mixed effects modeling probably is the most powerful
technique for analyzing experimental and observational data due to several
reasons. Mainly, the NM method does not share the drawbacks of the other
methods discussed above. Both stages of the TS method are performed in
one step, hence NM technique is also known as the one-stage method. One
of the chief advantages of the NM method is its ability to conduct meta-
analysis that is valuable in summarizing data across a drug development
program. The primary disadvantage of this method is the requirement of
sophisticated software that is not necessarily user-friendly for a wider
application. Usually, special training is required to use the software packages
and the learning resources are limited.
Model Qualification Methods
Model qualification is more popularly known as model “validation.” The

word “validation” implies a procedure of utmost robustness and may not be
applicable to the usual PK/PD models that are found in the literature.
Further, the fact that the true model and its parameters are not known
makes the choice of the word “validation” even poorer. A contrasting
example would be the validation of an analytical method, where “true”
concentrations of the chemical entity are known for making a calibration
standard. For wider acceptance, all models are required to be qualified and

236 Gobburu
credible. Clear specification of the purpose for which the model is being
developed is a prerequisite for any model building exercise.
Qualified Model/parameters. A model and its set of parameters are
deemed “qualified” to perform particular task(s) if they satisfy prespecified
criteria. Example: Application of posterior predictive check to a model and
its parameters for use in Monte Carlo simulations [4, 5].
Credible Model/parameters. A model and its set of parameters are
deemed “credible” [6] to perform particular task(s) if the conceptual
foundation on which the model was proposed is satisfactory to a group of
experts (subject matter-experts). Although there is no formal record of the
existence of such models, to the best of our knowledge, we speculate that (at
least the structural) models for warfarin [7] and reverse transcriptase/
aspartyl protease inhibitors [8] would be deemed as “credible.”
Monte Carlo simulations can be used to qualify a given model and its
parameters. Based on the objective, qualification methods can test either the
descriptive capacity or the extrapolation capacity of a given model.
Adequate description of the data will ensure that the proposed model and its
parameters are qualified to make inferences reliably within the range of the
data studied. Such a qualification will be assessed using the routine
diagnostic tests such as plots of the independent variable vs. observed and
(individual/population) predicted, summary statistics and determining the
precision of the parameter estimates. For example, developing an acceptable
descriptive model is critical for making labeling recommendations. Product
labels, usually, do not extrapolate results beyond the data range observed. A
model is qualified to predict beyond the range of the data used for building
the model if the descriptive capacity of the model is acceptable and the
model (and parameters, if applicable) is credible. It is important to note that
there is no means of assessing whether a model can be used for
extrapolation. Hence the credibility of the model i.e., whether the model
was derived from sound physiological principles and whether the submodel
and its parameters appear reasonable to a panel of experts, is important.
The guidance for industry on population pharmacokinetics presents a
variety of simulation methods that can be used to “qualify” models/
parameters [7]. Although a variety of methods for model qualification are
known, no thorough evaluation of their advantages and disadvantages is
available.
Model Based Dosage Optimization
Upon the selection of the appropriate PK/PD model, optimal dosage needs
to be derived for each patient. Two new “models” are introduced at this
point—the cost and the utility functions. The cost-utility analysis in PK/PD

modeling is relatively new and only the general theoretical principles will be
discussed here. The cost of a therapy can be defined as the “expense” of the
therapy due to an adverse effect, given the desired effect. Consider two
drugs—one for relieving migraine headache and another for treating
subarachanoid hemorrhage. Assume that both these drugs produce nausea.
Given the indication (migraine versus stroke), the cost of the two therapies
could be drastically different and hence may need different weighting. The
physician(s) and/or the patient decide the “cost” of a therapy, which makes
it highly subjective. The utility of a therapy can be defined as the advantage
the therapy is providing over not taking the therapy, given the cost of
declining therapy and the cost of drug-related toxicity.
Utility=f(Cost (No Therapy), Benefit, Cost (Toxicity) (3)
The utility function could have many components depending on the number
of desired and undesired effects. Figure 2 shows the concentration (or
dose)effect curves and the utility curve for various costs. Using the curves
such as those in Fig. 2, a target exposure and the region of therapeutic
equivalence should be determined. For example, the curve in Panel B for the
stroke drug suggests an optimal target exposure of about 100. Further, the
utility equivalence region would be, say, between 80 and 500% (asymmetric
FIGURE 2 The exposure (concentration or dose)-response (desired and

undesired) relationships of a hypothetical drug (Panel A). The utility of the therapy
was determined by subtracting the (cost adjusted) undesired effect from the
desired effect. The utilities of the therapy for two different desired effects [disease
reversal (stroke), migraine pain relief] given the same undesired effect (nausea) are
shown in Panel B. Note that declining therapy for stroke has a high cost. The
exposure that results in the maximum utility would be the optimal target exposure.
In Panel B, the optimal target exposure would be about 100 for the stroke drug and
zero for the antimigraine drug.

238 Gobburu
intervals). The corresponding exposures can then serve as surrogates for

individualizing drug exposures and establishing equivalence of two
products.
REGULATORY INITIATIVES
Several guidance documents for industry issued to date, reflect the leadership
of FDA in improving current drug development and in embracing good
scientific principles in the regulatory decision-making. Important messages to
industry, extracted from few guidance documents, are highlighted here.
International Conference on Harmonization of Technical

Requirements for the Registration of Pharmaceuticals for Human
Use (ICH) E4 [9]
The guidance for industry on dose-response information to support drug

registration states the use of a concentration-(desired/undesired) effect
relationship in individualizing therapy, optimal dosing regimen, and for
purposes of preparing dosing instructions in the product label. It further
notes that knowledge of the dose-response relationship enables multiple
regulatory agencies to make approval decisions from a common database.
Food and Drug Administration Modernization Act (FDAMA)
The implications of the FDAMA are discussed in the guidance for industry
on providing clinical evidence of effectiveness for human drug and
biological products [10]. Demonstrating effectiveness of a new drug product
usually requires more than one adequate and well-controlled investigation.
A full section entitled “extrapolation from existing studies” is devoted to
presenting a nonexclusive list of scenarios when additional clinical studies
are not necessary. The premise is that an acceptable benefit-risk ratio of a
drug product has already been established. Controlled clinical trials are not
necessary for approval of such a product for pediatric use and for
establishing equivalence of alternative formulations, modified-release
dosage forms, and different doses, regimens, or dosage forms. It is
important to note that the guidance emphasizes the availability of well-
defined concentration-effect relationships in the original new drug
application. The sponsors can very effectively take advantage of this
provision by prospective planning of the drug development programs.

Pediatric Exclusivity
The FDA offers a six-month extension of the patent on the use of a new drug,
should the sponsor fulfill the written request to characterize the PK/ PD of the
drug in pediatrics. As discussed in the above section, additional adequate and
well-controlled studies may not be required.
Recent Advisory Committee Meetings

The proceedings of two recent advisory committee meetings, one for the anti-
viral (AV) drug products and the other for the cardio-renal (CR) drug
products, are noteworthy. Both these meetings devoted 50% of the total
time to discuss the role of PK/PD in the AV and CR drug development. The
AV advisory committee discussed the role of modeling and simulation in
exploring various dosing regimens to appreciate resistance to the effects of
protease inhibitors over cumulative exposure and the importance of
compliance. The AV committee recommended that FDA should develop
guidance to the industry on the role of PK/PD in developing AV drug
products. The CR advisory meeting encompassed discussions on the need to
determine the exposure-response relationship. The FDA presented retro-
spective dose-effect analyses of more than 10 antihypertensive agents
previously approved over several years by the FDA [11]. The point that the
dose-response range of most of the drugs did not allow adequate
identification of the “optimum” dose was made. This affects several
regulatory decisions such as approval of combination drug products and
superiority claims. The outcomes of the meeting included: (1) use of
modeldependent analysis to learn about the shape of the exposure-response
curve and (2) need for more innovative designs that could potentially allow
frequentist and Bayesian types of data analysis.
Guidance to Industry on Population Pharmacokinetics [12]

and Exposure-Response
The guidance to industry on population pharmacokinetics emphasizes the
role of modeling and simulation [13] in designing trials and analyzing trial
outcomes. The exposure-response guidance focuses on the design and
analysis of data from studies characterizing the PK/PD of a drug.
The impact of the aforementioned regulatory recommendations issued by
the FDA is obvious. With efficient planning, sponsors can economize drug
development time and resources, and take full advantage of the incentives.
Building a concentration (not dose)-biomaker/surrogate/clinical endpoint
relationship during the development of a new drug for use in adults can
readily facilitate design (using simulations), analysis, and dosing

240 Gobburu
recommendations (labeling changes) for the drug’s use in pediatrics.

However, the ability of a concentration-effect relationship to support
approval of a dose/regimen not directly studied in clinical trials is not being
fully exploited. This is in fact one of the strongest uses of modeling and
simulation. Usually doses/regimens “directly” studied in clinical trials are
proposed in the labels. A model can effectively be used to explore the
suitability of intermediate doses not directly studied but could potentially
offer similar effectiveness as the other doses or dosing regimens.
Extrapolating outside the studied range may not be possible.
APPLICATIONS
Integration of Clinical Pharmacology Knowledge

The typical drug development strategies include dose ranging and bridging
studies. The dose ranging studies can be employed to model the
concentration (or dose)-effect (desired/undesired) relationships. The
clinical pharmacology characterization of a new drug involves a variety of
bridging studies to understand the influence of prognostic factors, such as
age, gender, smoking habit, food, hepatic/renal impairment, etc.
Effectiveness and safety data may not be collected in these types of studies,
but could be simulated from the previously developed model. A recent
example from a new drug application review is noteworthy. The dose-pain
relief (desired effect) and the concentration-heart rate (undesired effect)
relationship of a new drug, were both developed by meta-analysis of
various clinical studies.
In other studies, patients with severe renal impairment demonstrated a
60% decrease in the systemic clearance compared to that in normal subjects.
The influence of a 60% change in the drug exposure on effectiveness and
safety was simulated. Dosing without any adjustments in renal-impaired
patients causes negligible increase in the probability of pain relief and heart
rate. There is 100% probability that the increase in heart rate is within three
beats per minute. Whether a particular probability of occurrence of a given
magnitude of change, in the effectiveness and safety of drugs, due to
prognostic factors, is clinically relevant or not has to be mutually discussed
with the clinicians (domain-experts). The M&S offer a powerful method to
integrate knowledge across a submitted application. Simulating the
probability distributions of effectiveness and safety for the bridging studies
would enable a more informed and scientifically sound decision-making
regarding the necessity for a regulatory concern. Preserving and accessing
the knowledge when necessary at a later point of time will be much easier
and efficient. Further, such simulations can be instrumental in the

determination of exposure-equivalence intervals for the approval of changes

in the future formulations.
Special Populations
One of the most widely sought out labeling changes in special populations is
that for pediatrics. The application of M&S towards establishing in vivo
characteristics as a way to making labeling changes is worth discussing
further. The pediatric exclusivity policy is previously described (Sec. 2.3). If
there is reasonable belief that the disease process is similar in adults and
pediatrics and further an acceptable pharmacological effect marker is
available, then studies in pediatrics measuring the concentration-pharma-
cological effect(s) can be potentially used to recommend dosing changes in
pediatrics. The question that is being posed in the pediatric studies is: “Are
the pharmacokinetics/pharmacodynamics in pediatrics predictable from
those in adults?” Such a question can only be answered by developing
concentration-effect relationships. The sponsors are encouraged to employ
the model developed based on the PK/PD data in adults to design trials in
pediatrics. The analysis of the PK/PD data from trials in pediatrics may
require combining data from adults for a more complete understanding of
the drug behavior.
Influence of Prognostic Factors
One of the aims of modeling is to identify influential prognostic factors such

as body weight, age, gender, food, smoking habits, etc., on the fundamental
PK/PD parameters. Nisoldipine is formulated as a once-a-day controlled
release formulation of a dihydropyridine calcium channel antagonist which
is approved in the United States for the treatment of hypertension. Food was
found to increase the maximum concentration (Cmax: 2.75 vs. 7.5 µg/L)
and decrease the extent of bioavailability (AUC: 70.4 vs. 53 µg.hr/L) of the
controlled release product. The influence of the higher concentrations on the
decrease of blood pressure was evaluated using a previously developed
concentration-effect (blood pressure) model [14]. Simulations of the effect
under the Fed condition allowed in alleviating the safety concern of a large
drop in blood pressure. However in the labeling of Sular, administration on
an empty stomach for optimal bioavailability was recommended. The
docetaxel PK/PD relationship, in patients with cancer, was successful in
identifying a subpopulation, patients with liver impairment, to be more
prone to neutropenia (grade 4) [15]. This important finding was the basis
for the dosing recommendations in the labeling, for patients with liver
insufficiency. The drug development program of docetaxel exemplifies the

242 Gobburu
value added by the incorporation of prospective planning on the use of M&S

into the clinical trials.
FUTURE CONSIDERATIONS
M&S Team Structure/Communication
The biggest challenge, in the implementation of M&S projects, institutions

face today is team structuring and communication. Successful execution of
an M&S project undoubtedly requires teamwork and cooperation among
scientists from various disciplines (e.g., clinical, pharmacometrics, statistics)
and institutions (such as FDA and the industry). As aptly noted by Sheiner
[16], a clear definition of the roles of the “domain experts” (such as
clinicians/regulators) and “subject matter experts” (such as
pharmacometricians/statisticians) is the key to success and efficient
management of an M&S project. The domain experts would provide the
answers for the questions: (1) What do we want to know? (2) What are we
willing to assume, and (3) how certain do we need to be? Once the answers
for these questions are provided the subject matter experts will provide the
suitable experimental designs and analyses plans. It would take few
iterations to arrive at the final answers (which are in fact questions) and a
prospective design to achieve them. The M&S can be used as a very effective
tool during these “iterations.” Now, this exercise is particularly effective
when the discussions are between the regulatory agency and a drug sponsor.
The regulators will be in a position to comprehend “quantitatively,” the
rationale for the selection of a particular clinical trial design, in a timely
fashion. Further, the pharmacometricians and statisticians, who are the
designated “subject matter experts,” need to have a more active exchange of
knowledge across the two disciplines.
Pharmacometrics Training
The sources of learning pharmacometrics-related subject matter are very
limited. This situation needs to be addressed immediately for widening the
scope of M&S use. A pharmacometrician should have knowledge of basic
PK/PD concepts, adequate statistics background, good understanding of
physiological principles, and hands-on experience with at least one software
which can be used for M&S and another one to conduct statistical analysis.
Pharmacometricians also need to be trained in communicating “effectively”
with clinicians and statisticians. Regulatory agencies play a vital role in
emphasizing the importance of this discipline, as supported by the various
regulatory initiatives, discussed earlier. Industry should, then, recognize the

need for pharmacometricians and the academic institutions should train

them. A long-term solution, then, would be for the academic institutions to
offer graduate studies in pharmacometrics. A short-term solution is internal
training. The pharmacometricians within the institutions should venture in
collaborative projects thereby sharing the experience with the rest. Part of
the problem is also the practice of M&S as an art rather than a science.
Initiatives in streamlining the model-building process and making the
simulation exercise more transparent and reproducible are critical.
Time Intensity
Model building takes a longer time than performing and analyzing
simulations. Retrospective model building has two major steps—(1) data
access and (2) data analysis. The former is probably the rate-limiting step.
Typically, models are developed at the end of phase 3, most of the times. A
prudent way to economize time to develop models is by incorporating what
can be called as a “progressive model building (PMB) paradigm.” The
essence of the PMB paradigm is to update a model as new knowledge is
accrued. The PMB is advantageous because of at least two reasons. The first
one is being able to “carry-forward” the knowledge all along the drug
development for a given product and the second one is being able to divide a
big problem into several small components (“divide and conquer”) that are
easier to achieve. However, implementation of this paradigm calls for more
open collaboration of scientists from all disciplines and institutional
commitment to use the “current” model in designing the next trial. By
utilizing the PMB paradigm, scientists are almost forced to employ
mechanistic models, since the generalization power of empirical models is
limited. For example, it is much easier to update the parameter estimates of
an Emax model (with covariate effects) from a latest trial compared to those
of a cubic-spline model.
REFERENCES
1. Sheiner, L.B.; Ludden, T.M. Population Pharmacokinetics/Dynamics. Annu.

Rev. Pharmacol. Toxicol. 1992, 32, 185–209.
2. Sanathanan, L.P.; Peck, C.C. The Randomized Concentration-Controlled Trial:
An Evaluation of its Sample Size Efficiency. Control Clin. Trials Dec. 1991, 12
(6), 780–794.
3. Ebling, W.F.; Levy, G. Population Pharmacodynamics: Strategies for
Concentration-and Effect-Controlled Clinical Trials. Ann. Pharmacother. Jan.
1996, 30 (1), 12–19.
4. Gelman, A.; Meng, X.-L.; Stern, H. Posterior Predictive Assessment of Model
Fitness via Realized Discrepancies. Statistica. Sinica. 1996, 6, 733–807.

244 Gobburu
5. Gobburu, J.V.S.; Holford, N.H.G.; Ko, H.C; Peck, C.C. Model Optimization, via
“Lateral Validation” for Purposes of Clinical Trial Simulations. Clin. Pharmacol.
Ther. 1999, 65 (2), 164.
6. Law, A.M.; Kelton, W.D. Simulation Modeling and Analysis, 2nd Edition;
McGraw-Hill, Inc.; New York, 1991.
7. Nagashima, R.; O’Reilly, R.A.; Levy, G. Kinetics of Pharmacologic Effects in
Man: The Anticoagulant Action of Warfarin. Clin. Pharmacol. Ther. 1969, 10,
22.
8. Jackson, R.C. A Pharmacokinetic-Pharmacodynamic Model of Chemotherapy
of Human Immunodeficiency Virus Infection that Relates Development of Drug
Resistance to Treatment Intensity. J. Pharmacokinet. Biopharm. 1997, 25 (6),
713–730.
9. Guidance for Industry: Dose Response Information to Support Drug
Registration, http://www.fda.gov/cder/guidance/index.htm, 1999.
10. United States Food and Drug Administration Modernization Act 1997. http://
www.fda.gov/cdrh/modact97.pdf, 1997.
11. Gobburu, J.V.S.; Lipicky, R.J. Dose-Response Characterization in Current Drug
Development: Do We Have a problem? Part I: inferences from Animal/ Human
Data, http://www.fda.gov/ohrms/dockets/ac/00/backgrd/3656b2a.pdf, 2000.
12. Guidance for Industry: Population Pharmacokinetics; Center for Drug
Evaluation and Research, United States Food and Drug Administration, 1999.
13. Sun, H.; Fadiran, E.O.; Jones, C.D.; Lesko, L.; Huang, S.M.; Higgins, K.; Hu,
C.; Machado, S.; Maldonado, S.; Williams, R.; Hossain, M.; Ette, E.I.
Population Pharmacokinetics. A Regulatory Perspective. Clin. Pharmacokinet.
1999, 37 (1), 41–58.
14. Schaefer, H.G.; Heinig, R.; Ahr, G.; Adelmann, H.; Tetzloff, W.; Kuhlmann, J.
Pharmacokinetic-Pharmacodynamic Modelling as a Tool to Evaluate the
Clinical Relevance of a Drug-Food Interaction for a Nisoldipine Controlled-
Release Dosage Form. Eur. J. Clin. Pharmacol. 1997, 57 (6), 473–480.
15. Bruno, R.; Hille, D.; Riva, A.; Vivier, N.; ten Bokkel Huinnink, W.W.; van
Oosterom, A.T.; Kaye, S.B.; Verweij, J.; Fossella, F.V.; Valero, V.; Rigas, J. R.;
Seidman, A.D.; Chevallier, B.; Fumoleau, P.; Burris, H.A.; Ravdin, P.M.; Sheiner,
L.B. Population Pharmacokinetics/Pharmacodynamics of Docetaxel in Phase II
Studies in Patients with Cancer. J. Clin. Oncol. 1998, 16 (1), 187–196.
16. Sheiner, L.B. Dose Finding—What do We Want to Know? Cardiovascular and
Renal Drug Products Advisory Committee Meeting (FDA). Bethesda, 20
October, 2000.

12
Scientific and Regulatory Considerations for
Studies in Special Populations
INTRODUCTION
The course of development of an individual organism through successive

transformations in a lifetime is referred to as ontogeny. Consequences of
developmental changes and thus drug dosage modifications based on age,
liver function, renal function, and other intrinsic and extrinsic factors have
been well known for some time. Some examples of intrinsic factors are
genotype, gender, ethnicity, inherited diseases, acquired diseases, age specific
diseases, and polymorphism, and examples of extrinsic factors include
smoking, drug abuse, environmental pollutants, xenobiotic exposure, and
diet factors. During drug development it is not always possible to include
enough number of patients in pivotal clinical trials, to represent each
subpopulation. These subpopulations—also called special or specific
populations—include different ethnic and racial groups, age groups,
genders, pregnancy, lactation, and certain types of disease states (liver and
renal impairment) which may affect drug disposition, obesity, smokers, etc.
Pharmacokinetic (PK) and/or pharmacodynamic (PD) differences for all
245
246 Sahajwalla
these subgroups have been reported in the literature. In this book, pregnancy
and lactation have been discussed in Chapter 13, drug-drug interaction in
Chapter 14 and effects of certain disease states have been presented in
Chapter 15.
This chapter will introduce the readers to:
1. Some of the PK and/or PD differences reported for race, age,

gender, and obesity.
2. Regulatory perspective for gender, race, pediatric, and elderly
populations.
3. Study design considerations commonly used to assess differences
in specific populations.
4. Dose adjustment strategies.
As one can appreciate, this chapter is just an introduction to assessing

differences in important demographic subgroups and regulatory
perspective, it is not an extensive review and in no way a comprehensive
discussion of this vast field of special populations. Readers should also refer
to Chapter 2 of this book for regulations on special populations. The main
discussion in the following paragraphs will only focus on gender, race,
elderly and pediatric populations.
One of the major roles of clinical pharmacology is to provide information
which will aid in the individualization of the dose and dosing regimen. As
discussed later on, to identify when dosage adjustment may be necessary, it
is important to identify the limits of change in exposure of the drug that can
be accepted/tolerated for the drug being developed [1]. Once we have
identified the change in exposure that can be tolerated, one can recommend
adjusting the dose if that threshold has been reached in a specific
population, or in cases of drug-drug or drug-food interactions. Dose
adjustment strategies have been discussed later in the chapter.
DATA SUPPORTING THE NEED TO ASSESS DIFFERENCES IN

SPECIFIC POPULATIONS
Gender
Several examples have been reported in the literature that shows gender-
dependent pharmacokinetic and pharmacodynamic differences [2–21]. The
investigators have reported that the many differences in ADME based on
gender cannot be explained by differences in body weight or body
composition.

Scientific and Regulatory Considerations 247
Absorption of most drugs is a passive process and depends on factors

such as PKa, lipophilicity, and gastrointestinal physiology. Women secrete
less gastric acid and have slower gastric emptying than men. The mechanism
of this is unknown but has been hypothesized to be related to differences in
steroid hormone levels due to exogenous hormones and pregnancy [22, 23].
Gender specific absorption is rare and known examples are not found to be
clinically relevant [24, 25].
Distribution of drugs is influenced by physico-chemical properties,
vascular and tissue distribution, and ratio of lean body mass to adipose
tissue mass. Gender differences in drug distribution are related to body
weight and/or body fat proportion, whereas, plasma protein binding
differences are minor, and not of clinical significance [8, 19]. Many gender
differences are attributed to significant gender specific differences in drug
metabolism [15–17]. Total clearance of several CYP3A substrates appears
to be faster in women compared to men. Drugs metabolized by cytochrome
CYP1A, CYP2D6, CYP2E1, and Phase II metabolism such as
glucuronidation, conjugation, glucuronyltransferases, methyltransferases,
dehydrogenases, and by combined oxidative and conjugation processes are
usually cleared faster in men compared to women. Drugs metabolized by
CYP2C9, CYP2C19, and N-acetyltransferase, appear to have no gender effect
[3, 20]. Glomerular filtration, tubular secretion, and tubular reabsorption
appear to be faster in men compared to women [20]. Thus there are varying
degrees of gender-dependent clearance for several drugs. Some drugs are
cleared faster in females than in males, while some are cleared faster in males
than in females, whereas, many drugs have no gender-dependent differences
in their pharmacokinetics. Moreover, because of the difference in maturation
of each gender (for example, age at which puberty is reached), many
genderdependent pharmacokinetic characteristics of a drug may be
manifested as age-dependent factors [8].
The inclusion of women in clinical trials, and assessing gender differences
for the data obtained from pivotal clinical trials has been emphasized by the
FDA since two decades [27]. The Institute of Medicine has defined gender
difference as a difference between men and women due to cultural or social
variations in a particular sex. A sex difference has been defined as a
difference due to the sex chromosome or sex hormone [20]. The FDA has
described cultural, social, genetic, or hormonal differences between males
and females and used the term “gender differences” [2, 20, 21].
Literature has several excellent reviews summarizing the gender specific
differences in ADME and Pharmacodynamic variables. In general,
pharmacokinetic variability in gender has been better characterized
compared to pharmacodynamics variability. Limitations in measurements
of pharmacodynamic effects pose limitations (e.g., difficulty in quantifying
depression or perception of pain) [3]. Despite these limitations several

248 Sahajwalla
gender specific response data have been published [3, 4, 8]. Some of the
examples reported for pharmacodynamic differences include, women
having a better response to monoamine oxidase inhibitors (MAO) than to
tricyclics; more sensitivity to effects of ethanol; greater magnitude of
response to SSRIs, and; greater adverse events to cardiovascular drugs [8,
9, 20].
Race
The majority of literature information on PK and/or PD differences for race is

comparisons between Caucasians and Asians (often Chinese), and African
Americans and Caucasians. The influence of ethnicity on ADME
characteristics and PD of drugs have been reported and reviewed extensively
in the literature [29–39].
Drugs undergoing passive absorption are not expected to have any
differences. Calcium absorption is an active process and the fraction
absorbed in Caucasians is 25% vs. 44% in African Americans [29]. This
suggests that drugs undergoing active absorption may exhibit racial
differences.
Ethnic specificity in molecular genetics is one of the factors contributing
to the interethnic differences in drug disposition and response. The human
drug-metabolizing enzymes including CYP2D6, CYP2C9, CYP2C19,
CYP2E1, CYP2A6, aldehyde dehydrogenase (ALDH2), alcohol
dehydrogenase (ADH3) and non-P450 monooxigenase, N-acetyltransferase
(NAT2), glutathione S-transferase (GST), catechol-0-methyltransferase
(COMT), UDP-gucuronosyl-transferase (UGT), thiopurine methyltransferase
(TPMT), and dihdropyrimidine dehydrogenase (DPD), all display
polymorphism. Among these polymorphic enzymes, many of them had
exhibited known ethnic specificity including CYP2D6, CYP2C9, CYP2C19,
CYP2A6, UGT, NAT2, and ADH3 [40]. Further, gut metabolism via
CYP3A4 or PGP transport may affect absolute bioavailablity.
A review of 339 literature citations by Bjornson et al. [39] concluded that
no citation clearly described differences in active absorption of drugs
involving P-glycoprotein (PGP) transporters, α-1 Acid glycoprotein (AAG)
concentrations are reported to be lower in blacks and Chinese as compared
to Caucasians whereas, amounts of albumin are similar in these three
groups. Thus drugs binding exclusively to albumin are unlikely to show any
racial differences whereas, drugs binding to AAG are likely to have higher
binding, that is, a lower free fraction in Caucasians than in Chinese and
African Americans. However, none of the reported differences are clinically
relevant [39]. It may be advisable to assess race-dependent protein binding
especially for drugs predominantly bound to AAG. There is a potential for

race-dependent variability related to transporter [39]. Race-dependent

differences in metabolism are extensively reported. The incidence of poor
metabolizers of debrisoquinine phenotype in different populations for
CYP2D6 is, 7% for U.S. Caucasians, 0.7% for Chinese, and 0.5% for
Japanese, whereas, for CYP2C19 the incidence of poor metabolizers of
mephenytion is 3% for Caucasians, 17% for Chinese, and 22% for
Japanese. There are significant ethnic differences in enzyme activity of
CYP2C9, 2C19, 2D6, 1A2, 2A6, and N-acetyl transferase [39]. Based on in
vitro human liver microsomes of Caucasians vs. Japanese, 1A2, 2A6, 2D6,
2E1, and 3A4 enzyme activities are higher in Caucasians. Racial differences
in acetylators have been recognized since a long time. The frequency of slow
acetylators is as follows: African Americans 42–51%, Caucasians 52–58%,
Chinese 22%, Eskimos (Canada) 10%, and Japanese 7–12% [29]. Fifty
percent of Chinese and Japanese populations lack aldehyde dehydrogenase
enzyme activity resulting in accumulation of acetaldehyde which could
result in side effects like tacheycardia, palpitation, and facial flushing. In
summary, hepatic metabolism differences are the most common ethnic
differences.
Glomerular filtration and reabsorption being passive processes of
excretion are not likely to be affected by race. Tubular secretion is an active
process. In Chinese the renal clearance of metabolites of morphine is
significantly higher, suggesting that tubular secretion may be affected by
race [39].
The evaluation of drug response for several ethnic differences has also
been recently reviewed [39]. Some of the reported differences include—
African Americans having higher incidence of hypertension, interethnic
differences in vasodilatory response, and Chinese patients requiring a lower
daily dose of Warfarin. For drugs undergoing acetylation, populations with
a greater number of slow acetylators are likely to experience greater number
of adverse events. In addition to issues related to ethnic differences, other
factors such as diet, socio-economic status, exposure to environmental
pollutants, or interaction between these factors could play a role
contributing to ethnic differences, especially for the populations living in the
different regions of the world [41–48]. The effect of diet is not discussed in
this chapter, but has recently been reviewed in the literature [49].
Elderly
Elderly is defined as 65 years of age or older. Physiological changes occur in
aging which affect the ADME of drugs. The influence of age on
pharmacokinetics and pharmacodynamics has been extensively reviewed in
the literature [50–55]. In the elderly, the gastric pH is elevated, gastric
emptying time slightly reduced; intestinal motility, muscular blood flow,

250 Sahajwalla
plasma protein, and total body water are reduced; whereas, serum fatty acids
and adipose tissue are increased [50]. Kinirons and Crome [50] have
summarized the following accepted principles for elderly population:
decline in renal function with age, significant decline in liver size and mass,
significant reduction in hepatic blood flow; decreased cardiac output,
metabolic and renal clearance; in vitro content and activity of CYP450
enzymes or conjugation enzymes are not reduced with age. However, in vivo
clearance of drugs metabolized by CYP3A4, 2C9, 2C19, and 1A2 have been
reported to be reduced whereas, no reduction in clearance of drugs
metabolized by CYP2D6 and Phase II enzymes has been reported. With
regard to renal function, GFR, tubular secretion, and reabsorption are all
reported to be reduced in the elderly population. Differences in sensitivity to
drugs have also been reported with age for CNS and cardiovascular drugs
[50, 52].
Pediatrics
Children may exhibit different drug disposition and/or response compared

to adults. The pediatric patient cannot be considered as a “little” adult. It is
well documented that age-related developmental and physiological changes
exist not only in the pediatric population compared to adults but also within
pediatric age group. In addition, environmental (e.g., exposure to drugs in
vitro) and dietary factors can affect PK of drugs [56]. FDA guidance on
pediatrics and ICH E11 [57] define age groups within pediatric population.
The pediatric population is categorized into the following age groups—
preterm new born (gestation 23 to 34 weeks), term newborn infants (0 to 1
month), toddlers (1 to 24 months), children (2–11 years), and adolescents
(12–16/18 years).
Absorption of drugs can be affected by gastric pH, gastric emptying time,
and intestinal transit times. Gastric pH value is almost neutral at birth [6, 7]
then starts to vary from day eight and slowly declines to reach the adult
value by age three to seven years. This results in higher absorption of acid
labile drugs, such as penicillin and amoxicillin in toddlers and younger
children. Gastric emptying is prolonged until six months of age. Intestinal
transit time is decreased in children resulting in incomplete absorption of
sustained release products [58–61].
The total body water is increased and the percentage of body fat is
decreased in infants and children [62]. Albumin concentrations normalize
at one year of age and albumin binding is lower in infants. The
concentration of AAG is also higher over the first year [63, 64]. The
variability with age in these factors can affect drug binding and thus the
drug distribution [65, 66]. Further, the blood brain barrier in newborn

infants is not fully developed and drugs may cross the blood brain barrier
resulting in CNS toxicity [56, 67].
Both Phases I and II metabolizing enzymes are not mature at the time of
birth and different enzyme activity may reach the adult levels at different
ages (Table 1). For example, CYP3A4 activity may reach the adult level at
six months of age, whereas, CYP2D6 maturation occurs by five years and
CYP1A6 by 10 years of age. In case of renal excretion, the GFR, active
tubular secretion, and tubular reabsorption are lower in infants and nearly
equal to adults by 12 months of age and reach adult levels by childhood.
P-glycoprotein (PGP) expression has been associated with decreased gut
absorption of drugs and decreased amount of drugs crossing the blood brain
barrier. However, developmental aspects of PGP have not been investigated
[56]. Pharmacodynamic changes with age have been known for
neuromuscular blocking agents [68, 69].
Obesity
Recent reports indicate that obesity in the United States and worldwide is on
the rise [70]. Body mass index (BMI) is used to define obesity. Body mass
index is the ratio of the weight in kilograms to the square of the height in
meters [71]. The prevalence of childhood obesity has doubled in the last two
decades [72]. Estimates suggest that about 16% of children in the United
States may be obese. These estimates are higher in some minorities. Blounin
and Waren define obesity as a disease state characterized as a condition
from excess accumulation of body fat. Obesity is associated with changes in
plasma protein binding constituents and increase in adipose tissue mass and
lean body mass, organ mass, cardiac output, and splanchnic blood flow
relative to normal weight individuals [73].
Absorption in obesity is poorly understood, overall no significant
absorption differences in the obese compared to lean subjects have been
reported. For obese patients, drugs with less lipophilicity have little or no
change in VD. Increasingly lipophilic substances are affected by obesity.
Drugs predominantly bound to albumin do not show any significant
difference in protein binding [74–76]. AGP concentrations maybe higher in
obese patients resulting in decreased free fractions [77].
The effect of obesity on metabolism has not been well studied. The
activity of C4P3A4 is lower and that of CYP2E1 is higher in obese
compared to nonobese [78]. The effect of obesity on cytochrome P450 1A2,
2C9, 2C19, and 2D6 is inconclusive. Glucoronidation is significantly
increased and Sulfation may be moderately increased in obese [79, 80]. For
excretion, GFR has been shown to increase [81, 82] in some citations,
whereas it has also been shown to decrease [83]. This discrepancy has been

252 Sahajwalla
hypothesized to be due to different degrees of obesity in different studies.

Tubular secretion is possibly increased and tubular reabsorption is
decreased in obese [80, 84, 85].
Georgiadis et al. [86] assessed toxicity of several chemotherapeutic
agents to obese and compared toxicity to nonobese patients and concluded
that there was no correlation between toxicity and obesity. Each drug
behaved differently so predication of toxicity based on obesity was difficult.
Therefore, careful monitoring of narrow therapeutic index has been
recommended. When the same dose of triazolam [87, 88] was administered,
obese patients showed increased sensitivity. Desensitization of acetylcholine
receptors has been observed in obese [87].
With the incidence of obesity on the rise, it may become increasingly
important to assess obesity as a covariate during drug development.
REGULATORY PERSPECTIVE
Gender
In 1977, FDA issued a guidance which recommended that all women of
child bearing potential be excluded from clinical trials, unless adequate
safety, efficacy, animal fertility, and teratology information was available for
the drug being investigated [89]. This was done to protect the fetus, and the
assumption that men and women metabolize and respond to drugs in a
similar way [2]. In 1988, guidelines for the “format and content of the
clinical and statistical sections of the drug application” were issued which
required of the sponsors to discern dose-response relationships in the AEs
and examination of rates of AEs in various demographics (age, race,
gender) and other subgroups (metabolic status, renal function) [27]. In
1993, FDA revoked the 1977 guidelines and issued a guidance calling for
the inclusion of analyses of efficacy and safety data by gender, and
inclusion of characterization of pharmacokinetics of drugs in men and
women. The “Refuse to file” (RTF) guidance published by the FDA also in
1993, stated that NDA could be RTF if there was “clearly inadequate
evaluation for safety and/or effectiveness in the population intended to use
the drug, including pertinent subsets, such as gender, age, and racial
subsets” [90].
The U.S. FDA and other regulatory agencies have emphasized the need to
include subgroups such as gender, age, and race in the clinical trials. In order
to encourage recruitment of subgroups in clinical trails in all phases of drug
development, the Demographic Rule [91] was published in 1988, which
includes the following publications (2): for NDAs (21 CFR 314.50 (d)(5)(v)
and (d)(5)(vi)(a)); and for INDs (21 CFR 312.33 (a)(2)) and the clinical hold

rule. Guidance on Bioavailability and Bioequivalence issued by the FDA in

2000 also recommends that attempts should be made to include both sexes,
and representative ages and race. The International Harmonization
Conference (ICH) issued guidelines on clinical study reports (ICH E3) [92]
asking to include demographics and subgroup information to evaluate
safety and effectiveness in the subpopulations. It is evident that regulatory
agencies including ICH require inclusion of subgroups such as gender, age,
and race. The regulatory guidelines call for including enough number of
subjects to perform subgroup analysis.
Labeling for Gender

Summary of the 330 NDA reviews of drugs submitted between 1994 and
2000 [93] revealed that 163 drugs had gender specific information of which
122 drugs were new molecular entities (NME) and 39 of these drugs had
gender specific pharmacodynamics data. Eleven of these drugs were
identified as having greater than 40% differences in PK parameters. These
differences were described in the clinical pharmacology, special population,
or in the precaution sections of the drug labeling. Eight of the 39 drugs with
gender-related pharmacodynamic information, reported gender based PD
differences. Five reported increase in adverse events in females (neutropenia,
thrombocytopenia, QTC changes, risk of Torsade de Pointes, and other
mild adverse events), three drugs reported higher response in females
compared to males. These PD differences were not necessarily related to PK
differences. Of the eight drugs reporting differences in PD, five had less than
20% difference in PK parameters.
Toigo et al. [94] evaluated clinical review of the drugs approved between
1995–1999 to assess the participation of women in clinical trials and
gender-related labeling. Based on the review of clinical trial protocols and
labeling of 185 NMEs, they concluded that the participation of women in
clinical trials was proportionate to their representation in the U.S.
population. Labeling of 66% of drug products contained statements about
gender; only 22% described the actual gender effects. About 90% of the
gender effects discussed was PK related, 12% safety related and 5% efficacy
related. None of the labels recommended dosage adjustment for women.
Race
In 1985, the first regulation on special populations, 21CFR 314.5 asked for
evidence to support the dosage and administration section of label for
specific populations. In 1993, NIH published guidelines and they have been
updated in 2001 [95], which directed that appropriate proportions of
women and minorities be included in NIH sponsored clinical research.

254 Sahajwalla
These NIH guidelines called for review of the data to show whether
clinically important gender and minority based differences are expected. If
differences in response are expected then the phase III trial should be
designed to answer questions and include adequate sample size for
subgroups. The 1998 Demographic rule on IND and NDA requires that
Sponsors include analysis of effectiveness and safety, and modification of
dose and dosage regimen, for important demographic subgroups including
race (21 CFR 314.50 (d) (5) (VI) (a)). As stated in the section for gender
above, 21 CFR 314.10 (d) (3), FDA may refuse to file an NDA if pertinent
analysis for subsets of population is not included in the application.
International conference of Harmonization E5 (also printed at 63FR 31790,
June 1999) documents issued in 1998 describe the importance of evaluating
impact of ethnic factors on drug’s safety and efficacy. Since the ICH format
will allow the same application to be submitted in different regions of the
world it is important to evaluate the impact of ethnic factors, for
acceptability of data generated in foreign countries/populations. One of the
major issues in extrapolating clinical data from one region to another region
is the potential impact of ethnicity on the drug’s pharmacokinetics,
pharmacodynamics, drug efficacy, and toxicity [32].
To ensure consistency in subset analysis across studies, and to ensure
potential subgroup differences in a meaningful way, FDA is now
recommending [96] use of the standardized Office of Management and
Budget (OMB) race and ethnicity categories. This guidance recommends
that race and ethnicity information be a two-question approach and
subjects in a study self report that information. For ethnicity, two minimum
choices be offered, Hispanic or Latino, and Non-Hispanic or Latino. For
race the choices that be offered are American Indian or Alaska native, Asian,
Black, African American, Native Hawaiian or other Pacific Islanders, and
White. More detailed race and ethnicity information may be described but
the characteristics should be traceable to the five minimum categories
described above. Further, if studies are conducted outside the United States,
the race and ethnicity categories suggested in the guidance may not be
adequate to describe racial and ethnic populations in foreign countries.
Therefore, it is important that the information collected in foreign
populations be traceable to the recommended categories. The categories
recommended are the same as for U.S. population, with the exception that
the black or African American category can be replaced with a black or
African heritage category.
There have been several regulations recommending that the sponsor
include subgroup populations in the clinical development program. For
example, the Population PK guidance [97], Exposure-Response Guidance
[1], Content and Format of adverse reaction section of labeling for human
prescription drugs and biologies [98], clinical section of labeling [99], and

Best Pharmaceutical for Children Act, all ask for monitoring the race and
ethnicity of children participating in clinical studies. It is evident from the
regulations that are currently in place that regulatory agencies require
adequate participation and evaluation of racial and ethnic differences in
drug response.
Labeling for Race
Toigo et al. [100] reviewed 185 NMEs (approved for 1995 to 1999) for
participation of racial and ethnic subgroups in clinical studies. The review
findings were based on 2581 clinical trial protocols. They reported that
53% of clinical trial protocols had identified race. Whites represented 88%,
Blacks 8%, Asians pacific islanders 1%, and Hispanic Latinos 3%. For
Blacks the participation was consistent with the representation in the U.S.
population, while Hispanics appeared to be lower than their representation
in the U.S. population.
Review of these 185 drug labels [100] revealed that 84 (45%) had race
related statements. Fifteen of these labels contained 18 statements
indicating differences (9/18, 7/18, and 2/18, for PK, efficacy, safety
related, respectively) due to race/ethnicity. Ten, one and five product labels
were related to Blacks, Hispanics, and Asians, respectively. One
antihypertensive drug label recommended higher doses in Blacks based on
racial differences.
Elderly
As discussed above, ADME and pharmacodynamic response may be

affected with increase in age. To prevent or reduce the risk of adverse events
in the elderly, regulatory agencies have asked that the sponsors of new drugs
include sufficient number of elderly (65–75 years) and very elderly (greater
than 85 years of age) subjects in clinical trials. In 1977, the FDA established
the geriatric use subsection for labeling [101] to include information for the
elderly (21 CFR 201.57 (f) (10)) in the precaution section of the label. This
labeling regulation requires that all marketed drugs submit revised labeling
to include geriatric-use information. For details of this regulation refer to
the FDA website for relevant guidances. As stated earlier, the “Format and
content regulations” (63 FR 6854) require safety and efficacy data for
important demographic subgroups including age be included. IND
regulations (21 CFR 312.33 (a) (2)) require that annual reports by the
sponsors should contain the information on number of subjects enrolled in
clinical trials for certain subgroups including age. The “Content and forma
for geriatric labeling” guidance has been published in October 2001 and

256 Sahajwalla
gives a detailed procedure for submitting the “Geriatric Labeling

Supplement”. ICH guidelines also recommend inclusion and analysis of
data for elderly—ICH-E7, “studies in support of Special Population—
Geriatric”
Labeling for Elderly

To assess the availability of data on geriatric use in the label Sahajwalla and
Kwon (unpublished data) conducted a survey of 2002 Physicians Desk
Reference (PDR). A list of drugs was obtained by searching the key word
“elderly” in the electronic version of the PDR. Six hundred and fifty two
drug labels were listed with the key word “elderly,” eliminating different
dosage forms of the same drugs reduced this to a total of 549 drugs with
elderly information. The clinical pharmacology, precaution, and dosage
administration sections of these labels were reviewed. Out of 549 drugs, 141
drugs required dosage adjustments, 283 recommended cautions without
recommending a dosage adjustment, 103 did not require any dose
adjustments, and 22 drugs did not provide specific recommendation. Of the
141 drugs recommending dosage adjustment, 28 were based on PK findings,
100 due to PD findings, and 13 due to PK/PD findings. Forty one drugs
recommended decrease in the dose by 30 to 50%, and 10 drugs
recommended reducing the dose by more than 50%. Increased dosing
interval was suggested for four drugs and 82 drugs did not specify how
much dose reduction, but starting at a lower dose was recommended.
Caution for 263 drugs was advised in the label due to PK changes, increased
sensitivity, increased side effects, or the expected decreased renal, hepatic
and cardiac function in elderly. It is clear from these findings that during
drug development evaluating the effect of age on PK and PD of drugs is
essential.
Pediatric
The need for inclusion of pediatric information in the drug label has been
recognized by many drug regulatory agencies in the world. To encourage
pediatric labeling a final pediatric rule was issued by the FDA in 1994 [102],
which allowed adult efficacy data to be applied to pediatric patients with the
same disease or condition by supplementing and supporting the indication
with dosing and safety data in pediatric populations. In 1996, the content
and format for pediatric use supplement was issued [103]. In 1997, the Food
and Drug Modernization Act (FADMA) offered an incentive of six months
extension of exclusivity to market the drug product if studies were
performed in response to the FDA written request for pediatric studies.
Readers can refer to FDA guidelines on qualifying for Pediatric Exclusivity

under section 505(A) which was issued on June 30, 1998. In December
2001 FADMA expired, and in January 2002 the Best Pharmaceuticals for
Children Act went into effect, which provided similar incentives as the
FADMA. Other drug regulatory agencies in the world have also issued
guidelines to conduct studies in pediatric populations and to include these
populations in the product labeling. In August 1997, the Therapeutic
Products Directorate, Canada issued the “Inclusion of Pediatric Subjects in
Clinical Trials” guideline: in October 1997, the Australian Drug Evaluation
Committee issued a report of a working party on the registration of drugs
for use in children. In July 2000, ICH issued E 11 ‘Clinical Investigations of
Medicinal Products in Pediatric Population.’
In order to decide if only PK study with safety data is sufficient to support
pediatric indication or conduct of a PK and safety/efficacy trial will be
needed, a decision tree has been published in the FDA’s exposureresponse
guidance and presented below as Fig. 1.
FIGURE 1 Pediatric study decision tree.

258 Sahajwalla
STUDY DESIGN CONSIDERATIONS FOR SPECIAL

POPULATIONS
The goal of clinical pharmacology studies in special populations is to

determine how the dose and dosage regimen should be adjusted in special
populations so that the same systemic exposure that was found to be safe
and effective in the pivotal clinical trial for the population it was tested in
can be achieved. There are two approaches that can be adopted, a standard
PK approach and a population PK proach.
In a standard PK Approach, a single dose or multiple dose(s) of the drug
are administered (within the same study protocol) to the population being
investigated, e.g., males and females; different ethnic and race groups, adult
vs. elderly, and diffent age categories in the pediatric age groups. The
number of subjects included should be enough to obtain a reasonable
estimate of variability. Following the administration of the drug, frequent
blood and urine samples are collected and pharmacokinetic parameters
estimated and compared between the various populations of interest.
With the population PK (POPPK) approach, fewer samples are
collected from a larger number of subjects as compared to the Standard PK
approach, and PK parameters obtained are compared between the
populations of interest. The conduct of Population Studies is described in
Chapter 11 and in the FDA Guidance on Population PK [97]. Population
PK studies are generally conducted as an add-on study to Phase II and III
clinical trials. Some of the advantages of this approach include fewer
bloodsample collections. Thus ethical concerns of collecting several blood
samples from certain populations (e.g., pediatric) are reduced. The sample
collections can be part of a routine clinical visit when blood and urine are
being collected for other laboratory investigations. Since these studies are
generally being conducted as part of Phase II and III trials,
phramacodynamic endpoints can also be measured and exposure-response
(safety and efficacy parameters) relationships could be evaluated in
different populations of interest.
In order to decide which approach (standard PK vs. Population PK) is
better suited for conducting studies in special populations one should
consider the following. Regulatory agencies worldwide require the inclusion
of representative special populations in clinical trials, thus special
populations will be part of Phase II and III clinical trials. Therefore data
which can provide exposure-response (safety and efficacy parameters)
measures by including POPPK in the special population within pivotal
clinical trials would be more valuable than simply collecting information on
pharmacokinetic differences based on the standard PK approach. Based on
simulation studies some researchers believe that the population PK
approach is preferred over the traditional PK approach when characterizing

PK and PK/PD differences involving intrinsic (gender, race, age) factors. For
assessing the effect of extrinsic factors (different drugs, smoking, food, etc.)
one may not have enough subjects with the presence of that factor, enrolled
in clinical trails to assess differences based on POPPK.
DOSE ADJUSTMENTS
An important factor in deciding the dose adjustment is the knowledge of

exposure-response relationship [1]. Delineation of no-effect boundaries,
based on dose- and/or concentration-response studies would be beneficial.
Once the influence of intrinsic and extrinsic factors on drug exposure has
been characterized and exposure-response has been established, appropriate
dose adjustments can be recommended. Guidance on special populations
(hepatic, renal) and extrinsic factors (food effect, drug interactions)
recommend that in the absence of exposure-response data, the employment
of a standard 90% confidence interval of 80–125% for AUC and Cmax can
be used. If differences for populations of interest are within these boundaries
then dose adjustments are not needed. These guidances also acknowledge
that “FDA recognizes that documentation that a PK parameter remains
within an 80–125% no effect boundary would be very difficult given the
small numbers of subjects usually entered into these studies. If a wider
boundary can be supported clinically, however, it may be possible to
conclude that there is no need for dose adjustment.”
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Supplements” May 1996. http://www.fda.gov/cder/guidance/index.htm

13
Conducting Clinical Pharmacology Studies in
Pregnant and Lactating Women
Kathleen Uhl
INTRODUCTION
Pregnant and lactating women are two special populations that present
unique challenges for conducting research. Many women of reproductive
age group (15–5 year) may have chronic medical problems and use a variety
of pharmaceutical products (e.g., drugs, vaccines, and other biologic
therapies). In the U.S., 60 million women are of reproductive age (15–44)
[1], and there are about four million births per year [2]. The magnitude of
major chronic conditions in women less than 45 years is significant. In this
population, asthma affects 6,099,000 women; epilepsy affects 466,000; and
hypertension affects 2,700,000 [2]. The prevalence of these conditions
among pregnant women are 7% for asthma, 0.6–1.0% for epilepsy, and 6%
for hypertension [2]. Thus, many women enter pregnancy with medical
conditions that require ongoing or episodic treatment. New medical
problems may develop or old ones may be exacerbated by pregnancy (e.g.,
infections, migraine headaches, depression). Lactating women, as well, may
require medication for chronic or acute conditions.
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268 Uhl
Pregnant and lactating women are usually not part of the traditional drug
development program. As a matter of fact, pregnant and lactating women
are actively excluded from most clinical studies. If pregnancy does occur
during a clinical study, treatment is discontinued and the patient is frequently
dropped from the study. Consequently, at the time of initial marketing, except
for products developed to treat conditions specific to pregnancy (e.g.,
tocolytic agents for preterm labor, treatment of preeclampsia), there are
usually no data on the appropriate dosage and frequency of administration
during pregnancy. The same situation may also be seen after years of
marketing; data in product labels regarding pharmacokinetics and dose
adjustments during pregnancy and lactation rarely provide more information
than was available at the time of initial marketing.
Decisions can and should be made during drug development to study the
kinetics of products in these subpopulations. If the drug is anticipated to be
used by women of reproductive age, then developers should consider when
and how to study pregnant and lactating women because the drug will be
used by them once marketed. If a drug has a good maternal- and fetalsafety
profile, studies can be performed in pregnancy. Pharmacokinetic/
pharmacodynamic (PK/PD) studies in pregnant and lactating women should
be considered if the drug is prescribed in or used by pregnant and lactating
women or pregnancy or lactation are likely to significantly alter the PK of a
drug (e.g., effect of pregnancy on a drug that is renally eliminated). These
studies are especially important if use of the drug would be required and not
optional to treat maternal medical conditions. If there is no systemic
exposure to the product, or the product is not used by women of
childbearing age, during pregnancy, and lactation there may be no need to
conduct PK/PD studies.
The medical literature provides information about drugs being used in
pregnant and lactating women and should help investigators select products
for further study. Information on human pregnancy and lactation exposures
and experiences usually emerge during the postmarketing phase for
pharmaceutical products. Postmarketing data that demonstrate fetal and
maternal safety help reduce the obstacles to performing PK studies in
pregnancy. Publications in the medical or lay press may describe use of a
drug in pregnancy and medical specialty groups may publish position
statements or clinical recommendations for specific drug therapy for clinical
scenarios. Publications may describe safety or efficacy in lactating women,
safety in the breast-fed child via exposure to drug in breast milk, case
reports describing use of a drug in lactating women, and information from
medical specialty groups (e.g., consensus documents or opinion papers).
These sources can help with determining the research questions to be
investigated, and will additionally be useful when designing a protocol and
informed consent documents, and obtaining IRB approval.

Conducting Clinical Pharmacology Studies 269
Health care providers and their patients must make decisions about the
use of medications during pregnancy and lactation with little to no data to
guide them in decision-making. The ultimate goal of PK/PD studies in
pregnant and lactating women should be to provide meaningful information
for patients and their health care providers so that they can make informed
decisions about drug use and appropriate dosing during pregnancy and
lactation. Studying Pharmaceuticals in pregnancy and lactation requires
special considerations including methodological design, data analysis, and
ethical and regulatory considerations. When studies are performed in
pregnant and lactating women, frequently the study utilizes only a few
women. In addition, methodologies are often inadequate to draw
substantial conclusions and have little influence on clinical prescribing
scenarios.
This chapter will address considerations for investigators who recognize
the importance of drug use in pregnant and lactating women, the need for
data to assist prescribing, and despite the obstacles, choose to study
pregnant and lactating women.
PREGNANCY
Introduction
Although the ideal situation during pregnancy is abstinence from the use of
pharmacologic agents, many women use prescription or over-the-counter
drugs during pregnancy. Several studies have shown that pregnant women
do use prescribed or over-the-counter drugs during pregnancy [3–5]. A
survey of approximately 20,000 women over a 25-year period (1976–2000)
demonstrated that drug (excluding vitamins and minerals) use in pregnancy
is increasing [6]. The mean number of drugs women reported using during
pregnancy over this 25-year period has increased from 1.7 to 2.9. Over 80%
of all women reported using any drug during pregnancy, and approximately
30% reported using>four drugs. In addition, of the top 10 reported drugs
used, six were over-the-counter (OTC) products. In Europe a comparison of
therapeutic drug use during pregnancy showed that 64% of women used at
least one drug during pregnancy [4]. In France, pregnant women were
prescribed an average of five drugs during the first trimester [5].
Physiology of Pregnancy
Pregnancy is a dynamic state of altered physiology. The physiologic changes

inherent to pregnancy can affect the pharmacokinetics and/or
pharmacodynamics of drugs (Table 1).

270 Uhl
TABLE 1 Physiologic Changes in Pregnancy with Potential to Alter ADME
Absorption Delayed gastric emptying

Prolonged gastrointestinal transit time
Distribution Decreased gastric acid secretion, higher gastric pH
Increased plasma volume
Increased extracellular fluid
Increased total body weight
Decreased plasma albumin
Respiratory alkalosis
Increased cardiac output
Metabolism Increased estrogens and progesterone
Decreased CYP1A2 activity
Increased CYP3A4 activity
Increased CYP2D6
Elimination Increased renal blood flow
Increased glomerular filtration rate
Increased creatinine clearance
Some physiologic changes are abrupt while others evolve more slowly
during pregnancy. Most of the physiologic changes manifest during the first
trimester and peak during the second trimester of pregnancy. Obstetric
textbooks provide a more elaborate discussion of the physiology of
pregnancy. Briefly, pregnancy causes changes in total body weight and body
fat composition. Pregnancy may affect the bioavailability of drugs because
gastric emptying is delayed [7], gastrointestinal transit time is prolonged [8],
and gastric acid secretion is decreased [9]. Plasma volume expands during
pregnancy with significant increases in extracellular fluid space and total
body water that vary with patient weight and can affect the volume of
distribution of drugs [10]. Hemodynamic changes in pregnancy include an
increased cardiac output, increased stroke volume, and elevated maternal
heart rate. Blood flow to the uterus, kidneys, skin, and mammary glands is
increased. The percent of cardiac output attributed to hepatic blood flow is
lower in pregnancy than that in the nonpregnant condition [11]. The
concentration of plasma albumin decreases during pregnancy resulting in
reduced protein-binding [12]. Glomerular filtration rate increases early in
pregnancy and continues to rise throughout pregnancy [13]. Hepatic
enzyme activity has also been reported to change during pregnancy,
including CYP450, xanthine oxidase, and N-acetyltransferase [14, 15].
Physiologic changes are not fixed throughout pregnancy but rather reflect a
continuum of change as pregnancy progresses.
The multiple physiologic changes in pregnancy provide the rationale for
investigating the pharmacokinetics and pharmacodynamics during

pregnancy. However, despite the altered physiology the assumption is often

that the pharmacokinetics in pregnancy are no different from healthy
volunteers and pregnant women are dosed similarly. Unfortunately there is
little information available to direct appropriate prescribing for pregnant
women. In the absence of information the usual adult dose is prescribed in
pregnancy and may result in substantial underdosing or excessive dosages.
Scientifically driven dosing recommendations derived from well-designed
and well-conducted PK/PD studies are critical to the health of the pregnant
woman and potentially the fetus.
Sources of Information Regarding Drug Use in Pregnancy
Before any investigator pursues studying drug kinetics in pregnancy,

information regarding drug safety of that particular product will be crucial
to designing a protocol and subsequently obtaining Institutional Review
Boards (IRB) approval. Even though information in product labeling is
usually limited, multiple other sources are available that provide
comprehensive information that assess reproductive toxicities from drug
exposures. For example, the on-line REPRORISK system available from
Micromedex, Inc. contains electronic versions of four teratogen information
databases: REPROTEXT, REPROTOX (www.reprotox.org), Shepard’s
Catalog [16], and TERIS [17]. These periodically updated, scientifically
reviewed resources critically evaluate the literature regarding human and
animal pregnancy drug exposures. Other sources of information are the
more than 20 comprehensive multidisciplinary Teratogen Information
Services (TIS) located in the United States and Canada, which provide
patient counseling and risk assessments regarding potential teratogenic
exposures (www.otispregnancy.org). Many TIS, such as MotherRisk
(www.motherisk.org), employ genetic counselors, who are excellent
resources for pre- and postconception counseling.
Of the thousands of pharmaceutical products available only a handful
are known human teratogens [18]. Largely as a result of the
thalidomideinduced birth defects, most people, both patients and clinicians,
over-estimate the risk to pregnancy from drug use and perceive it to be quite
large [19, 20]. The overall incidence of major malformations in the general
population has been estimated at 1–5% [17]. The etiology of most
congenital malformations remains uncertain; approximately 20% are
caused by genetic factors and chromosomal abnormalities and 10% are
caused by environmental factors such as maternal conditions (4%),
infections (3%), and chemicals and drugs (approximately 1% or less) [18].
Teratogenicity is only one important aspect of drug use in pregnant women;
however, the appropriate dose necessary for anticipated efficacy is critical as

272 Uhl
well. Sources of information on appropriate dosing in pregnancy are not

available.
Methodologic Considerations
The ultimate goal of studies performed to determine the effect of pregnancy
on PK/PD should be to provide useful information for appropriate dosing of
drugs in pregnancy. A well-conducted study begins with a well-designed
study. Studies in pregnancy may require extensive collaborative efforts that
enlist the support of specialists in obstetrics, pediatrics, pharmacology,
pharmacometrics, and statistics, among others.
Study Objectives
The protocol should clearly state the primary objective of the study, e.g., to
determine the PK and/or PD in pregnant patients, or to determine if the PK/
PD are altered in pregnant patients to such an extent that the dosage should
be adjusted.
Study Participants and Control Group

The study participants optimally should be representative of the typical
patient population for the drug to be studied. Consideration should be
carefully given to the control group selected, and the study protocol should
provide the rationale for the control group selected (Table 2).
For PK studies in pregnancy, PK parameters should optimally be
compared in the pregnant and nonpregnant state with the woman serving as
her own control by undergoing serial PK/PD assessments. This type of
design will avoid the criticism of some PK/PD studies of pregnant women
which are flawed by the comparison group selected [21, 22]. Ideally PK
assessments would be done prepregnancy for baseline PK and in all three
trimesters, although this is rarely practical. For chronically administered
drugs an assessment of prepregnancy PK/PD could be done. When the
patient becomes pregnant and if her medical condition requires that she stay
on the drug of interest and the drug has a good fetal-safety profile, PK/PD
assessments during pregnancy could be compared with prepregnancy. A
study center that enrolls patients on chronic therapy for medical conditions
prior to pregnancy would be best suited for this study design.
Many pregnant women do not seek obstetric medical care until the end of
the first trimester, therefore, it may be very difficult to enroll pregnant
women in the first trimester. Practical considerations limit most PK studies
to the 2nd and 3rd trimesters with the baseline assessment done in the
postpartum period. If the study design is such that each woman serves as her

Conducting Clinical Pharmacology Studies
TABLE 2 Participant and Control Group Options and Sampling Strategies
+
lmmediate assessments at 24–48 hours postpartum.
*Remote assessments at ⱖ2–3 months postpartum.
#
Pop PK studies do not need to use the same patient in sequential sampling time frames.
273
274 Uhl
own control, PK/PD should be determined during the postpartum period

and ideally this would include an early or remote (or both) postpartum PK/
PD determination. The remote assessment should take place at least 2–3
months postpartum to allow for the physiologic changes inherent in
pregnancy to return to the nonpregnant state. In addition, the women
should not be lactating for the postpartum assessment to best reflect the
nonpregnant state. Sometimes pharmacologic therapy needed during
pregnancy will no longer be necessary in the postpartum period (e.g.,
hypertensive medications to control pregnancy-induced hypertension). If a
drug possesses linear kinetics a single-dose postpartum PK/PD study could
be extrapolated to multiple dose steady-state kinetics during pregnancy.
Consideration should be given to the inclusion and exclusion criteria and
must be tailored to the study taking into account the drug and/or the disease
being studied. Factors with significant potential to affect the PK/PD of a
drug (e.g., the trimester of pregnancy, age, weight, diet, smoking, alcohol
intake, concomitant medications, ethnicity, renal function, other medical
conditions) may need to be considered as well. Uniform diagnostic criteria
should be applied across pregnant patients to ensure similarity of diagnosis
and also minimize drug-disease interactions that could contribute to
variability. The study protocol should include the criteria for dating the
pregnancy and this should be consistently applied (e.g., using last menstrual
period or ultrasound for dating the pregnancy). The metabolic status should
be considered for drugs that are hepatically metabolized and known to
exhibit genetic polymorphisms (e.g., CYP2D6 or CYP2C19). Genotype has
been shown to have an affect on pregnancy-related changes in metabolism
[15].
Pharmacokinetic/pharmacodynamic studies could also be nested within a
larger clinical study on safety, efficiacy, or pregnancy outcomes. For
example, the PK of nifedipine was studied in a small subset of patients who
were participating in a larger clinical study to assess treatment for
pregnancy-induced hypertension [23].
As discussed earlier, the physiologic changes in pregnancy are dynamic
and continuous throughout pregnancy and are not necessarily imminent
with each trimester. In order to minimize variability for traditional PK
designs, investigators should consider narrowing the time of sampling from
a trimester of gestation to a “window” of gestational age. For example, the
protocol could prospectively state “windows” of time for study, e.g., 20–24
weeks instead of any time in the 2nd trimester.
Sample Size
The determination of an adequate sample size depends on the objective and
design of the study. Considerations for sample size should include the PK

and/or PD variability for the drug being studied, the study design (i.e.,
single-dose vs. multiple-dose), and the variability of the physiologic changes
inherent in pregnancy. Intraindividual and interindividual variabilities may
differ in pregnancy compared with the nonpregnant state and should be
considered when determining the sample size. For a population PK
approach, sparse sampling with a larger number of patients may be useful as
well [24].
The final number of patients enrolled may need to be in excess of the
sample size calculated to take into account drop-outs or subsequent patient
exclusion from the study, especially for longitudinal study designs. Some
patients may be excluded from study participation in a subsequent trimester.
Data for that patient will be missing for the trimester of interest; however,
the patient should be continued in the study so that postpartum PK/PD
assessments are done.
Sample Collection and Analysis

Consideration should be given to the type (e.g., plasma, whole blood, urine)
and number of samples that are necessary to accurately estimate the relevant
pharmacokinetic parameters for the parent drug and its active metabolites.
Since plasma protein binding is often altered in pregnancy, total and
unbound concentrations of drug and metabolites should be determined.
Unbound drug concentrations are generally believed to determine the rate
and extent of delivery to the sites of action. For drugs and metabolites with
a relatively low extent of plasma protein binding (e.g., extent of binding less
than 80%), alterations in binding due to pregnancy are most likely small in
relative terms.
Data Analysis
The analysis of the study will depend on the study design characteristics.
Total and unbound plasma drug/metabolite concentrations (and urinary
excretion data, if collected) can be used to estimate PK parameter. The PK
parameters can include the area under the plasma concentration curve
(AUC), peak concentration (Cmax), plasma clearance (CLT) or the apparent
oral clearance (CL/F), apparent volume of distribution (Vz/F or VSS/F), and
terminal half-life (t1/2). Pharmacokinetic parameters should be expressed in
terms of total and unbound concentrations. For drugs and metabolites with
a relatively low extent of plasma protein binding (e.g., extent of binding less
than 80%), description and analysis of PK in terms of total concentrations
are usually sufficient. Noncompartmental- and/or compartmental-modeling
approaches to parameter estimation can be employed.
Mathematical models for the relationship between pregnancy status and
relevant PK parameters can be constructed. The categorization of

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gestational age, either as a nominal (e.g., trimester) or a continuous (week of

gestation) variable will direct the appropriate type of analysis. The analysis
may provide an estimation of PK/PD parameters, modeling of the PK/PD
relationship, and modeling of the relationship between gestational age and
the PK parameters. The models selected should be adequately supported by
the data and/or mechanistic arguments.
In addition, an assessment of whether dosage adjustment is warranted in
pregnant patients and recommendations for dosing can be further
extrapolated. Typically the dose is adjusted to produce a comparable range
of unbound plasma concentrations of drug or active metabolites at baseline
(prepregnancy or postpartum) compared to that during pregnancy.
Simulations may identify doses and dosing intervals that achieve the goal for
pregnant patients at different trimesters or gestational ages. Special
statistical considerations may be necessary for longitudinal study designs
given the repeated measures characteristics of the design.
Study-Design Considerations
A longitudinal study design should be considered for drugs that are
administered chronically or given for several treatment cycles throughout
pregnancy. In this design, pregnant women would have pharmacokinetic
assessments conducted serially throughout pregnancy and each woman
would then serve as her own control. The study should focus on comparing
a pregnant patient at one trimester of pregnancy to the same patient at a
different trimester as well as to the same patient at baseline (prepregnancy
or postpartum). This type of design could potentially minimize
interindividual variability throughout pregnancy.
It may be difficult to use a longitudinal study design for drugs that are
given acutely (e.g., single dose or short course of therapy) in pregnancy. In
such cases, a multiple-arm study design could compare different pregnant
patients at different trimesters, e.g., a sample of women each in 2nd and 3rd
trimesters. Each woman could again serve as her own control and have PK/
PD determinations performed in the postpartum period. If it is impossible to
administer drug to the same patient in the postpartum period, then an
additional arm of the study using a different population of postpartum
women, or female volunteers, could be used.
Ideally, the dose given for a PK/PD study in pregnancy should reflect
actual clinical usage. If the drug is usually given chronically during
pregnancy, multiple dosing for steady-state kinetics would be optimal. In
some circumstances, the dose may need to be increased or decreased as
pregnancy progresses, to achieve the appropriate therapeutic response, e.g.,
lowering of blood pressure, or to decrease, adverse events such as
hypotensive episodes with antihypertensive therapy. In designing the study,

investigators should consider how changes in dose over pregnancy will be

handled in the analysis.
A population PK study design may also be considered. A particular
advantage of the population PK approach is the assessment of multiple
covariates. Techniques such as nonlinear mixed effects modeling may be
used to model the relationship between covariates such as gestational age
and PK parameters such as the apparent clearance of the drug (CL/F). The
control group selected for a population PK study design may differ from
other designs and may be normal female volunteers [8].
Drug Metabolism (CYP450) Studies

Drug metabolism studies using probe substrates have been performed in
pregnant women [14]. One concern about the use of probe substrates in
pregnancy is the lack of direct therapeutic benefit to the pregnant woman or
her fetus. For drug metabolism studies, a single dose of a probe substrate
could potentially be given during pregnancy although there may be
circumstances that limit dosing probe substrates in a pregnant woman. It
may be reasonable to administer a probe substrate once or twice during
pregnancy and once in the postpartum period for each woman in order to
minimize nontherapeutic exposure to a drug. Alternatively, lower doses of
probe substrates can be used in pregnancy studies.
Pharmacodynamic Assessments
Whenever appropriate, pharmacodynamic assessment should be considered
when designing PK studies in pregnancy. The selection of the PD endpoints
should be carefully considered and may be based on the pharmacological
characteristics of the drug and metabolites (e.g., extent of protein-binding,
therapeutic index, and the behavior of other drugs in the same class in
pregnant patients). Similarly, biomarkers may be considered to measure PD
endpoints of interest. Consideration should also be given to fetal PD
assessments, e.g., fetal heart rate and rhythm response to maternal
administration of an antiarrhythmic drug.
Ethical Considerations and Regulatory Framework

Ethical Considerations
Ethical considerations for studying drugs in pregnant women must be
tended to in the study design and when conducting studies. Some
recommend that only pregnant women who require a drug for therapeutic
reasons be included in clinical studies, citing that drug studies cannot be
done in normal pregnant “volunteers” [25]. Others believe that women

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should already have made the decision to use the particular drug of interest
to treat a medical condition during pregnancy in order for a study to
proceed. The patient should not, ordinarily, be making the decision to take
the study medication in order to participate in the study. Drugs can be
studied for maternal medical treatment (e.g., hypertension, seizure disorder)
as well as for fetal treatment (e.g., fetal tachycardia).
Protection of Human Subjects Regulations

Studies that are supported by federal funding must comply with 45CFR46,
Protection of Human Subjects [26]. Subpart A of this regulation is the basic
Department of Health and Human Services Policy for Protection of Human
Research Subjects, and contains basic protections for human research
subjects participating in clinical research. Expedited review for studies that
represent minimal risk to study subjects is possible under this regulation.
Federal regulations require that IRB give special consideration to protecting
the welfare of particularly vulnerable subjects, such as children, prisoners,
pregnant women, mentally disabled persons, or economically or education-
ally disadvantaged persons. Institutional Review Board approval is
necessary and ensures that risks are minimized and reasonable with benefits
to subjects of study participation. Institutional Review Boards’ ensure that
subject selection is equitable, require informed consent for studies, review
protocols to ensure safety and subject confidentiality, and ensure protection
of vulnerable subjects. Many IRBs follow federal regulations on the conduct
of studies in pregnant women.
Subpart B of this regulation, modified in 2001, is critical to conducting
studies in pregnant women and contains additional protections for human
fetuses, pregnant women, and human in vitro fertilization (Table 3).
According to Subpart B, pregnant women can give informed consent and
engage in research studies if (1) studies have been conducted on animals and
nonpregnant women; (2) research meets the health needs of the mother and
the risk to the fetus is the minimum necessary or minimal risk; and (3)
research benefits the mother, fetus, or general knowledge. In general,
maternal consent is all that is necessary for the participation of pregnant
TABLE 3 Protections of Human Subjects

Regulations Pertaining to Pregnant Women
Benefits of study Consent required
General knowledge Maternal only

Maternal health Maternal only
Fetal health Maternal & paternal

women in studies. However, for studies that benefit only the fetus, both
maternal and paternal consent are required for maternal participation in
such studies.
Regulatory Requirements
Studies conducted under an Investigational New Drug (IND) application or
with federal financial support must comply with 45CFR46 with specific
attention paid to Subpart B regarding paternal consent and with
21CFR312. Studies done to support a labeling claim should comply with
ICH E6, The Good Clinical Practice: Consolidated Guideline [27]. “Positive
or negative experiences during pregnancy or lactation” will be one safety
issue to be explicitly addressed in the Overall Safety Evaluation section of
the Periodic Safety Update Report (PSUR). The International Conference on
Harmonisation Guidance for Industry E2C Clinical Safety Data
Management: Periodic Safety Update Reports for Marketed Drugs [28]
contains more information regarding these regulatory submissions. This
requirement will eventually be incorporated into the FDA Safety Reporting
Regulations. Postmarketing exposure and safety data will most likely
provide the appropriate background that supports the need for
pharmacokinetic assessment in pregnant patients.
Incorporating PK/PD Data in Pregnancy Labeling

The current regulations regarding pregnancy labeling (21CFR 201.57 (6)(a)-
(e)) promulgated in 1979 use the pregnancy categories (A, B, C, D, and X) to
address teratogenic risk to the fetus from drug exposure (Table 4).
TABLE 4 U.S. Food and Drug Administration Pregnancy Labeling Categories
Pregnancy
category Category description
A No adverse effects in humans.

B No effect in humans with adverse effects in animals OR
No effects in animals without human data.
C Adverse effects in animals without human data OR
No data available for animals or humans.
D Adverse effects demonstrated in humans OR
Adverse effects in animals with strong mechanistic expectation of effects in
humans.
X Adverse effects in humans or animals without indication for use during
pregnancy.

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Prior to 1979, there was no requirement to address pregnancy in labeling.

The current regulations address decision-making for the use of drugs by
women who are already pregnant.
The newly proposed physician labeling rule [29] describes “pregnant
women” as a special population. Unless a product has been specifically
studied for an indication unique to pregnancy (e.g., treatment of preterm
labor), treatment during pregnancy is not considered an “indication” for
regulatory purposes. Rather, pregnant women are considered a
subpopulation with altered physiology. Erroneously many health
professionals and the medical literature discuss the use of drugs in
pregnancy as “indicated for” or, more typically, “not indicated” for
pregnancy.
Information from PK/PD studies in pregnancy should be included in
product labeling. The labeling should reflect the data pertaining to the effect
of pregnancy on the PK and/or PD (if known) obtained from studies
conducted. Information from these studies may need to be cross-referenced
to other labeling sections such as the clinical pharmacology, special
populations, warnings, precautions, pregnancy, and dosage and
administration sections. The FDA is working to improve the quantity and
quality of data available on the use of medications during pregnancy and is
in the process of revising the pregnancy labeling regulations to delete the
pregnancy categories scheme and promote more useful clinical information
in a narrative format [30–33].
LACTATION
Introduction
Breast milk is widely acknowledged to be the most complete form of

nutrition for infants. Breastfeeding poses multiple benefits for infants
including health, growth, immunity, and development. Specific infant
benefits of breastfeeding include decreased episodes of diarrhea, respiratory
infections, and ear infections. Breastfeeding poses multiple maternal
benefits as well, including a reduction in postpartum bleeding, earlier return
to prepregnancy weight, reduced risk of premenopausal breast cancer, and
reduced risk of osteoporosis [34]. In order to encourage breastfeeding, the
Health and Human Services “Healthy People 2010” initiative targets
increasing the percentage of mothers who breastfeed to 75% in the early
postpartum period, 50% at six months, and 15% at one year [35].
Professional medical organizations encourage breastfeeding as well [36, 37].
The American Academy of Pediatrics (AAP) considers breastfeeding to be

the ideal method of feeding and nurturing infants and recommends that all
women breastfeed and continue to do so until the child reaches one year of
age [37].
As in pregnancy, it is highly likely that a woman will require and take
medications while she is breastfeeding. Surveys in various European
countries demonstrate the extent of drug use by lactating/breastfeeding
women. Postpartum women who choose to breast feed take fewer
medications than those who do not breastfeed [38]. Most nursing mothers
(90–99%) receive a medication during the first week postpartum, 17–25%
of nursing mothers take medication at four months postpartum, and 5% of
nursing mothers receive long-term drug therapy [39].
When lactation studies are undertaken, the emphasis is usually on the
health risk or extent of exposure in the breast-fed infant, failing to
investigate maternal factors such as pharmacokinetics, dose adjustments, or
other clinically relevant information that affect the efficacy or safety in
breastfeeding women. Potential differences in PK might be expected in the
postpartum and lactating periods due to differences in endogeonous
hormones, total body weight, body fat, and muscle mass compared to
nonlactating women.
Inconsistent and inadequate methodologies are often employed in
lactation studies. Many studies have shortcomings such as an extremely
small sample size with infrequent or single-time point sampling, thus
making interpretation or comparison across studies quite difficult. The
consistent application of adequate study designs should improve both the
quality and quantity of data available, and assist patients and health care
providers when making decisions about the use of drugs in lactating
women.
The mere presence of a drug in breast milk does not necessarily indicate a
health risk for the breast-fed infant. The presence or absence of the drug in
milk is only the first step in determining risk. The extent of exposure to a
drug in the breast-fed infant may be considerably less than anticipated by
drug excretion into breast milk due to decreased bioavailability of drug in
milk (e.g., tetracycline). In addition, the known or anticipated effects on the
breast-fed child of drug exposure through breast milk will aid in the risk
analysis. Unwarranted recommendations to stop nursing will negate the
benefits of breastfeeding to both the mother and the child.
Clinical lactation studies can be designed to address different lactation
issues such as PK/PD changes in lactating women, extent of drug transfer
into breast milk, extent of drug transfer via breast milk to the breast-fed
child, drug effect on milk (e.g., production and composition), and effects of
drug exposure from breast milk on the breast-fed child. This section
addresses considerations in the design of clinical lactation studies. The

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design for safety studies in the breast-fed child specifically studying the effects
on the breast-fed child of drug exposure through breast milk is beyond the
scope of this section.
Physiology of Lactation
Lactation is an integral part of the reproductive cycle of humans. Breast

development begins in utero; however most of the morphogenesis of the
breast occurs postnatally in adolescence and adulthood. Under the influence
of sex steroids, especially estrogen, the mammary glandular epithelium
proliferates. The breast is prepared for milk production during pregnancy
through the complex endocrine changes of pregnancy, especially prolactin.
Lactogenesis, the initiation of milk secretion, has been described as a three-
stage process [40]. Stage I begins approximately 12 weeks before delivery
and is marked by increases in lactose, proteins, including immunoglobulins,
and decreases in sodium and chloride. Lactogenesis is initiated after delivery
with a fall in serum progesterone, and high prolactin levels. The first milk
secreted is called colostrum. This initiation of lactogenesis in Stage II does
not rely on infant suckling until the third or fourth postpartum day. In Stage
II, the blood flow to the breast increases. Oxygen and glucose uptake by the
breast increases as does the citrate concentration. At days two and three
postpartum, Stage II becomes clinically apparent with copious secretion of
milk typically referred to as “the milk coming in.” Major changes in milk
composition continue for approximately 10 days, usually referred to as
“transitional milk” and then “mature milk” is established; this final stage of
lactogenesis is referred to as Stage III. The process of milk secretion requires
milk synthesis and milk release.
Human milk differs from milk of other species in that the concentration
of monovalent ions is lower and lactose is higher [41]. Milk contains over
200 constituents and is isosmotic with plasma. Lactose is the major
carbohydrate for the milk of most species and is only found in milk. Breast
milk is high in lipid most of which is long-chain fatty acids. Most proteins in
milk are formed from free amino acids in the secretory cells of the breast and
are specific to breast secretions [42]. Human milk contains up to 4000 white
blood cells/mL and is particularly high in colostrum. Macrophages are the
white blood cells found in greatest number. Mature human milk has a pH
that is more acidic than plasma [43]. Human milk is not a uniform fluid but
one of changing composition [44]. Milk composition differs within a given
feeding with foremilk differing from hindmilk, e.g., fat content is highest in
hindmilk. Colostrum differs from transitional and mature milks. Milk
composition varies with maternal nutrition, the time of day, and among

women [43]. Drugs can potentially alter the composition of breast milk
including changes in protein, lactose, lipid, and electrolyte concentrations
[45].
During the weaning process when milk is not removed or is less
frequently removed, the increased pressure in the breast decreases blood
flow and inhibits lactation. Milk protein, chloride, and sodium
concentrations increase and lactose concentrations decrease during
weaning. Involution of the mammary gland occurs when regular extraction
of milk from the breast ceases and involves an orderly sequence of events
[43]. Involution is characterized by secretory epithelial cell apoptosis,
degradation of the mammary gland’s basement membrane [46], and gland
remodeling reverting to the prepregnant state. Involution is accompanied by
a decrease in the activity for most of the enzymes involved in lipid synthesis
[47]. It is not known exactly how long it takes for a lactating woman to
return to her baseline status (e.g., nonpregnant, nonlactating state) after
weaning is complete.
Sources of Information about Drug Transfer into Breastmilk
It is generally believed that all drugs pass into breast milk. Drugs pass into
milk by simple diffusion, carrier-mediated diffusion, or active transport.
Factors that influence the amount of drug that passes into breast milk
include the molecular weight, protein-binding, degree of ionization,
solubility, both lipid and aqueous, and the pH of plasma relative to breast
milk.
There are a number of articles of drugs in breast milk including reviews
and studies of a specific medication. The AAP has published consensus
documents listing drugs and chemicals that are transfered into breast milk
[48–50]. These publications include recommendations about drug use
during breastfeeding as well. In addition, textbooks and other references are
available that provide information about the use of specific drugs in breast
feeding, including data of safety and drug transfer into milk [51, 52].
Many references include the milk/plasma ratio (M/P) for many drugs as
an estimate of the dose of maternal drug delivered to the infant via
breastmilk. The M/P ratio is the concentration of drug in the milk vs. the
concentration of drug in maternal plasma (or serum). Pitfalls exist in the
estimation of the M/P ratio, the most common of which is the assumption
that milk and plasma drug concentrations parallel each other throughout
dosing [53]. Presumed concurrence between milk and plasma drug
concentrations weakens the reliability of reported data, as do M/P ratios
reported from single time point determinations. PK studies in lactation must

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account for the time-dependent variation of drug concentration in milk and

plasma.
Considerations for Conducting Clinical Lactation Studies

Clinical lactation studies may be undertaken to investigate PK/PD changes
in lactating women. Lactation studies could also investigate the extent of
drug transfer into breast milk and subsequently the extent of drug transfer
into the breast-fed child. In addition, lactation studies could be designed to
investigate alterations to breast milk from maternal drug exposure, such as
milk volume and composition. This type of study could be done for drugs as
well as larger biological molecules, especially if there is the potential to alter
the composition of breast milk, e.g., vaccines and altered immunologic
properties of breast milk. Finally, clinical lactation studies can be designed
to investigate the effects on the breast-fed child from drug exposure via
breast milk. There are many areas to consider when designing clinical
lactation studies.
Methodologic Considerations
Several publications have addressed the methodologies for conducting

studies on drug transfer into breast milk. A World Health Organization
(WHO) Working Group published guidelines for conducting studies on the
passage of drugs into breast milk [39, 54]. In addition the environmental
health community has substantial experience in assessing exposures through
breast milk. Some of the methodologies used in environmental health
studies may be useful when designing human studies to assess exposures to
Pharmaceuticals through breast milk. The WHO European Centre for
Environmental and Health has been involved with monitoring
environmental exposures via studies on levels of chemicals in human milk,
particularly polychlorinated biphenyls (PCBs), polychlorinated dibenzo-
pdioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) [55]. An
expert panel discussion provided recommendations for developing a breast
milk monitoring program for environmental exposures in the United States
[56]. This report includes recommendations for participant selection,
methods for obtaining human milk, detecting the presence of environmental
chemicals in those samples, and interpreting and communicating the
information found.
Study Objective
The primary objective of the study in lactating women should be clearly
stated, for example, to determine if the PK and/or PD are altered in lactating

women such that dose adjustment is necessary. Careful consideration should

be given to adequate baseline determinations and comparisons to baseline.
For example, for studies that are conducted to evaluate the effect on milk
production (e.g., the quality or quantity of breast milk), the diurnal
variation of milk production and composition should be considered in study
design. Study design (e.g., participant selection, number of study subjects,
sample collection) will vary according to the primary study objective.
Study Participants and Control Group

Study participants may include mother-infant pairs or lactating women
alone. Optimally, the study participants would be representative of the
typical patient population for the drug to be studied. Maternal factors with
significant potential to affect lactation (e.g., weight, gravity, parity, stage of
lactation, postpartum status, episodes and duration of previous
breastfeeding) or the PK of a drug to be studied (e.g., diet, smoking, alcohol
intake, concomitant medications, ethnicity, other medical conditions)
should be considered. Inclusion and exclusion criteria should be carefully
considered and need to be tailored to the study. Infant factors (e.g., age, term
vs. preterm neonates, extent of breastfeeding, and age related changes in
absorption, distribution, metabolism, and excretion) should be considered
as well. Uniform diagnostic criteria should be applied to all patients to
ensure similarity of diagnosis for which treatment is being given to reduce
disease-specific variability in PK.
Careful consideration should be given to the control or comparison
group chosen. For clinical studies, ideally the lactating woman would serve
as her own control by undergoing PK/PD assessment(s) in lactation and
again after weaning is complete, e.g., a longitudinal study design. The
optimal control group will depend on the research question asked and the
objective of the study. Potential control groups include historical controls
(usually male volunteers) or female volunteers with or without the medical
condition of interest. If female volunteers are used as controls, consideration
should be given to matching them to study subjects (e.g., postpartum status,
age). The control group should account for postpartum PK changes and
identify time windows (e.g., 3–4 months postpartum) to account for
variability in physiologic postpartum changes. The post weaning samples
for PK/PD should be performed at similar times after weaning as well, e.g.,
one month after weaning in complete. The rationale for the control group
selected should be provided in the study protocol.
Sample Size
Determination of an adequate sample size depends on the objective and
design of the study. The number of patients enrolled in the study should be

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sufficient to detect clinically significant differences (e.g., PK differences large

enough to warrant dosage adjustments). The PK variability of the drug as
well as the PK/PD relationships for both therapeutic and adverse responses
will affect this decision. Sample size considerations should include PK and
PD variability for the drug being studied, the study design (i.e., single-dose
vs. multiple-dose), and the variability in lactation physiology. Inter and
intrasubject variability for mother and breast-fed child may need to be
considered depending on the design and primary objective of the study. A
population PK design could also be considered however practical difficulties
in conducting a population PK study during lactation may limit its value.
The final number of patients enrolled may need to be in excess of that
originally calculated by standard sample size calculations and should take
into account drop-outs and subsequent exclusion from the study.
Sample Collection and Analysis

The frequency and duration of sampling should be sufficient to accurately
assess the outcome selected, e.g., estimate the relevant pharmacokinetic
parameters for the parent drug and its metabolites (see Data Analysis
section below). Samples should be collected in a manner to characterize the
complete dosing interval. Each breast should be completely emptied at each
sampling time, the volume of milk recorded, and an aliqout removed for
analysis. An electric milk pump is recommended since milk composition can
vary with the method used. Separate collection containers should be used
for each milk collection. Pooling of different-timed milk samples is not
recommended. Consideration should be given to sample handling and the
protocol should include the precise details especially with milk samples (e.g.,
methods to minimize contamination). Total and unbound concentrations of
drug and metabolites should be determined. Bioanalytical methods should
determine drug and metabolite concentrations in all biological matrices
studied (e.g., plasma, serum, whole blood, breast milk, urine). Milk samples
should additionally be assayed for milk fat.
Data Analysis
Total and unbound plasma and milk concentration data (and urinary
excretion data, if collected) can be used to estimate PK parameters of the
parent drug and metabolites concentrations. Maternal PK parameter
estimates can include: the area under the milk concentration curve (AUCm
or AUCmilk; AUC0–t or AUC0–∞ in single dose studies and AUC0–τ at steady
state), the area under the plasma concentration curve (AUCp or
AUCplasma; AUC0–t or AUC0–∞ in single dose studies and AUC0–τ at steady

state), peak concentration (Cmax), time to peak plasma concentration (tmax),

plasma clearance (CLT) or the apparent oral clearance (CL/F), apparent
volume of distribution (V Z/F or V SS /F), and terminal halflife (t 1/2 ).
Pharmacokinetic parameters should be expressed in terms of total and
unbound concentrations. For drugs and metabolites with a relatively low
extent of plasma protein binding (e.g., extent of binding less than 80%),
description and analysis of PK in terms of total concentrations are usually
sufficient. As warranted by the study conducted, infant PK parameter
estimates could be determined. The PK parameters of metabolites in
maternal plasma, breast milk and ingested by the breast-fed infant can be
estimated. If samples obtained from the breast-fed infant do not permit
determination of both total and unbound (e.g., insufficient number and
volume of samples), the average fraction of drug bound can be determined.
Noncompartmental and/or compartmental modeling approaches to
parameter estimation can be utilized.
The amount of drug or metabolite consumed by the breast-fed infant, the
daily infant dosage, can be determined. The amount of drug excreted in
breast milk over 24 hours was chosen arbitrarily since it represents a single
day of exposure to drug via breastmilk. Any time frame could be chosen,
e.g., dosing interval; however, it may be easier to interpret daily results.
The infant dosage can be calculated by summing the product of drug
concentration and the volume of milk obtained at each sampling time
interval:
Daily infant dosage (mg/day)=Σ(total drug concentration in each milk

collection time interval×expressed milk volume in each milk collection
time interval)
Alternatively, the infant daily dose can be estimated with the following
equation:
Estimated daily infant dosage (mg/kg/day)=M/P×average maternal

serum concentration×150 mL/kg/day
where M/P (milk-to-plasma ratio) is the ratio of AUCmiik to AUCplasma, the

average maternal serum concentration refers to AUC0–∞/dosing interval after
maternal ingestion of a single dose of drug or AUC0–τ/dosing interval at
steady state during chronic maternal dosing [39, 54]. Calculation of the M/
P ratio from single paired maternal milk and plasma concentrations
obtained at one sampling time is not recommended because it fails to take
into account the time-dependent nature of the M/P ratio [53, 57]. The
standardized milk consumption of 150 mL/kg/day, the mean milk intake of
a fully breast-fed two-month-old infant, is used [39, 54, 57, 58].

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If infant dose is calculated by both the above-mentioned methods, these

data should be compared and explanations sought for disparities in results.
Subsequently, the percent of the weight-adjusted maternal dose
consumed in breast milk over 24 hours can be calculated:
% Maternal dosage=(Infant dosage (mg/kg/day)/Maternal dosage

(mg/kg/day))×100
Similarly, this could be calculated for a dosing interval. If the pediatric or

infant dose is known (i.e., the drug is approved for pediatric use), the
percent weight adjusted pediatric dose ingested can be estimated as well.
The infant serum concentration is probably the most direct measure of
infant risk from a drug received from breast milk. If infant serum data are
not collected, the average infant serum concentration (C ss,ave) can be
estimated by:
Css,ave=F×infant dosage/CL
where F is the bioavailability and CL is the drug clearance in the infant, if

the data are known for the pediatric population.
When studying drugs during lactation the investigator must consider the
balance and relationship between mother, breast milk, and the breast-fed
child. The optimal study would evaluate all three components (e.g.,
mother—infant pairs); however, in some circumstances other designs can be
useful (e.g., maternal milk) and may need to be performed before a mother-
infant pair study is conducted. Other potential designs include only those
lactating women studies which provide data on the PK of the drug in
lactating women and the amount of drug transferred into breast milk.
Alternatively, only women studies that provide data exclusively on milk may
verify other studies (e.g., in vitro data) that predict drug transfer in human
milk. In some circumstances the study of milk alone may preceed more
intensive investigation utilizing mother-infant pairs.
In general mother-infant pair studies should measure the amount of drug
and metabolites transferred into breast milk, characterize the PK of the drug
in lactating women, and assess drug exposure in the breast-fed child via
breast milk. This design would include frequent maternal blood and milk
samples that are simultaneously obtained and carefully timed. This design
would also include infant sampling of blood and/or urine and would
encourage alternative noninvasive pediatric sampling strategies (e.g., saliva,

tears) to reliably determine drug levels and PK parameter estimates in

infants.
Clinical lactation studies could be nested within a larger clinical study on
safety or efficiacy outcomes or conducted in combination with the
postpartum assessment of the effects of pregnancy on PK/PD of a drug.
Information obtained from single-dose studies are useful and may be more
acceptable to volunteers and aid in recruitment; however, the normal
therapeutic practice (e.g., dose, frequency, and route of administration)
should be considered in the study design. When drugs are normally taken in
repeated doses, studies performed at steady state are encouraged. For probe
substrates for drug metabolism studies drugs a single dose could be given.
As with pregnancy study designs, a multiple-arm design could be used.
For drugs that are given acutely (e.g., single dose or short course of therapy)
it may be difficult to use a longitudinal design with the same patients
throughout lactation. If there is a concern that the effects of drug use in
lactation differ based upon the stage of lactation, or the postpartum status,
a multiple-arm design could be considered. Each woman could serve as her
own control and have PK/PD determinations performed once during
lactation and after weaning is complete.
Pharmacodynamic Assessments
Whenever appropriate, pharmacodynamic assessment should be included in
clinical lactation studies. The selection of the PD endpoints should be based
on the pharmacological characteristics of the drug and metabolites (e.g.,
extent of protein binding, therapeutic index, and the behavior of other
drugs in the same class in lactating patients). Similarly, biomarkers could be
used to measure PD endpoints of interest. Consideration should be given to
PD assessments in the breast-fed child as well, e.g., heart rate and rhythm
response to maternal administration of drug.
Ethical Considerations and Regulatory Framework
Ethical Considerations
Ethical considerations for studying drugs in lactating women must be
tended to in the study design and when conducting studies. Since clinical
lactation studies that do not expose the breast-fed infant to drug can be
done, usually the ethical hurdles are not as problematic as with pregnancy.
In general, if breast-fed infants are included in clinical lactation studies,
women should already have made the decision to use the particular drug of
interest to treat a medical condition during breastfeeding and have made the
decision to continue to breastfeed in order for a study to proceed. The

290 Uhl
patient should not, ordinarily, be making the decision to take the study
medication in order to participate in the study.
Protection of Human Subjects Regulations

As with studies in pregnancy, lactation studies that are supported by federal
funding must comply with 45CFR46, Protection of Human Subjects, and
should have IRB approval. Investigators participating in studies that involve
breast-fed infants should be familiar with Subpart D of this regulation
regarding requirements for permission by parents or guardians
(45CFR46.408) for infant participation in clinical studies.
Regulatory Requirements
A Nursing Mothers section is required in labeling (21CFR 201.57 (f) (8));
however, there are no regulations requiring that studies be performed in
lactating women. The Agency has provided guidelines for the study of
gender differences and states that it is medically important that a
representative sample of the entire population likely to receive the drugs has
been studied [59].
Labeling
As with pregnancy, the newly proposed physician labeling rule [29]
describes “Lactation” as a special population; lactating women are
considered a subpopulation with altered physiology. When available,
information from clinical lactation studies is often included in product
labeling. Information from these studies may need to be cross-referenced to
other labeling sections as well. Simply indicating that “drug is present in
breast milk” or reporting the M/P without the contextual setting are not
very helpful for patients or prescribers. Labeling should provide clinically
meaningful information to assist health care providers and their patients
make decisions about drug use in lactation.
AREAS FOR FURTHER RESEARCH
Clinical pharmacology and PK studies in pregnant and lactating women can

identify factors that affect drug PK, such as maternal characteristics (e.g.,
age, gravity/parity, race, weeks gestation), concomitant medications, or
underlying medical conditions. Studies can also serve as hypothesis
generating tools for further study.
In the past, stable isotopes have been used extensively for intrinsic
metabolic studies; however, their use in pharmacologic studies, especially in

pregnant or lactating women, is limited. The metabolism of glucose during

pregnancy has been studied using stable isotope labeled glucose [60–63].
The idea of using an intravenous dose of a stable isotope labeled drug
administered simultaneously with an unlabeled oral dose of the same drug
to determine bioavailability was first introduced in 1975 [64]. No studies
using stable isotopes in human pharmacologic studies have been published
since 1989; however, a few investigators advocate the use of stable isotopes
as a means to determine absolute and relative bioavailability in pregnant
women [24, 25, 65, 66]. Studies employing stable isotopes present some
potential advantages over traditional PK approaches and would decrease
the number of studies necessary, decrease the biologic variation between
studies (intraindividual variability), and decrease sample volume.
In addition, physiological-based PK (PBPK) modeling in animals has been
utilized to predict drug transport across the placenta [67]. This type of
modeling may have applicability for human pregnancy, however, animals
typically used in such modeling have substantially shorter gestations
compared with humans. Human pregnancy is more complicated and PBPK
models designed for human pregnancy may be extremely complex.
Modeling may only predict passive transport across the placenta, failing to
take into account active transport processes. Physiological-based PK
modeling could be further developed and validated to predict maternal PK
changes resulting from pregnancy-induced physiologic changes. In vitro,
animal or human placental models are useful to help predict if a drug is
transferred across the placenta, as well as the extent of drug transfer, and the
mechanism of transfer.
Non-clinical models (e.g., mechanistic, in vitro, animal, physicochemical-
based, and PBPK) can predict the amount of drug in breast milk and may be
applicable to predict infant exposures to drug in breast milk as well. The
applicability and validity of nonclinical models to human lactation is still
under investigation. Data obtained from clinical lactation studies can test
the predictive value of the nonclinical models. The incorporation of the
additional information obtained from clinical lactation studies into
nonclinical models should improve the predictability of the nonclinical
approaches.
New technologies for studying drug disposition may be particularly
valuable in investigating gender differences in PK/PD and pharmacogentics
[68]. The correlation between genetics and phenotype of drug effect in
pregnancy and lactation requires further investigation and may be useful in
the accurate prediction of clinical outcomes. Chronopharmacology,
including chronopharmacokinetics and chronopharmacodynamics, may be
important in pregnancy and lactation studies. The integration of complex
information about genotype, phenotype, circadian effects, and other
outcomes requires sophisticated databases, and database development may

292 Uhl
serve as powerful adjuncts that allow for exploration of the relationships

among complex variables.
CONCLUSIONS
Many challenges are met when studying special populations such as renal or
hepatically impaired patients; however, studying pregnant and lactating
women presents some unique challenges. Pharmacokinetic and pharmaco-
dynamic studies in pregnant and lactating women can assist in providing the
appropriate dosage and frequency of administration in pregnancy and
lactation and optimize the efficacy and safety of these products. Information
drawn from scientifically conducted PK/PD studies will hopefully assist
health care professionals and their patients in decision-making about the use
of medications during pregnancy and lactation.
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14
Scientific, Mechanistic and Regulatory Issues
with Pharmacokinetic Drug-Drug Interactions
Patrick J.Marroum
Hilde Spahn-Langguth
Martin-Luther-University
Halle-Wittenberg
Wolfgang-Langenbeck-Str., Germany
Peter Langguth
Johannes Gutenberg-University
Germany
INTRODUCTION
A drug interaction implies a likely modification of the expected response to
the drug in an individual, due to the exposure of the individual to one or
more drugs or substances. Drug interactions which produce adverse
reactions in patients are unintentional, yet drug interactions may also be
intentional if they provide an improved therapeutic response or allow for a
more convenient dosing regimen [1]. Drug interactions include drug-drug
interactions, food-drug interactions and chemical-drug interactions, such as
the interaction of a drug with alcohol or tobacco.
297
298 Marroum et al.
In general, the frequency of possible drug interactions increases with the

number of concomitantly administered drugs, multiple prescribers, poor
patient compliance, patient risk factors such as predisposing illness, or
advancing age. Several of these factors are interrelated. Elderly patients and
patients with chronic illnesses such as hypertension or diabetes are on
multiple drugs. Recent estimates show that hospital patients are
concomitantly administered 7 to 12 drugs thus rendering the clinical
outcome of such polypharmacy difficult to predict. Furthermore, the clinical
significance and severity of a potential interaction needs to be estimated
(major, intermediate, minor). For example, the interactions between
ketoconazole and terfenadine, cholesterol-synthesis (CSE) inhibitors (e.g.,
lovastatin, simvastatin), or pimozide are being classified as major drug-drug
interactions due to the foreseeable side effects and the limited therapeutic
range of the drugs involved. In the case of terfenadine or pimozide
administered together with imidazol or triazol antimycotics, a prolongation
of the QT-interval, ventricular tachycardia (Torsades de pointes) with loss of
consciousness, and perisystole have been reported [2]. A combination of
ketoconazole or itraconazole with CSE-inhibitors may result in severe
myalgia and myopathia and may ultimately lead to rhabdomyolysis, a loss
of skeletal muscle mass. On the other hand, the combination of
ketoconazole with Cyclosporin A and certain benzodiazepines (e.g.,
midazolam, triazolam) has been categorized into the intermediate severity
class. In the case of Cyclosporin A therapeutic drug monitoring and
monitoring of kidney function has been recommended, whereas with
oxidatively biotransformed benzodiazepines, a reduction of their dose needs
to be considered or alternatively, a benzodiazepine which is not eliminated
by oxidative biotransformation is recommended. The decrease of the
bioavailability of ketoconazole by concomitant administration of H2-
antihistamines has been termed a minor interaction [2]. This interaction is
due to the dependence of dissolution of ketoconazole upon gastric pH and
an increase in gastric pH will ultimately lead to a reduction of the
dissolution rate of ketoconazole. This interaction can be avoided, if the H2-
antihistamines are dosed two hours before or six hours following the dosing
of ketoconazole.
This chapter provides an overview of the different mechanisms by which
pharmacokinetic drug-drug interactions occur and an overview of the
regulatory considerations with regard to the study of drug-drug interactions
from the U.S. Food and Drug Administration, the European and the
Canadian health authorities’ perspectives. Finally, the role of the population
screen in the study of possible drug interactions in phase III clinical trials
will be briefly outlined.

Scientific, Mechanistic and Regulatory Issues 299
DRUG-DRUG INTERACTION MECHANISMS
Pharmacokinetic drug-drug interactions are commonly classified according

to whether they occur during the absorption, the distribution, the
metabolism, or the elimination phase (ADME). An alternative—
mechanistic—classification scheme groups drug-drug interactions into:
i. drug-drug interactions based on the reaction with one or more

macromolecules
ii. physicochemical interactions and interactions based on changes in
local pH and, connected therewith, changes in the ionization state
of molecules
iii.based on pharmacodynamic mechanisms.
Drug-Drug Interactions with Involvement of Macromolecules

Drug-drug interactions with the involvement of macromolecules are based
on either the blockade of binding sites of one drug by a competing drug, or
generally, the change in binding behavior of a drug to a macromolecule in
the presence of an interacting molecule, or a change in the amount of
macromolecules present (e.g., an increase of drug metabolizing enzymes in
the presence of enzyme-inducing drugs).
Macromolecules that are important contributors of a drug-drug
interaction can be drug-metabolizing enzymes, which catalyze phase I or
phase II metabolic reactions, resulting in the formation and elimination of
pharmacologically active and/or inactive metabolites. Furthermore, a drug-
drug interaction can take place as a result of an interaction of drugs with
one or more, transporter proteins, which may be critical for the passage of
drugs across biological membranes. This process is sometimes also being
referred to as phase III of drug metabolism. In this particular case, the
excretion of a polar—membrane impermeable—metabolite from the
intracellular compartment in which it has been formed, is enhanced by
binding to and subsequent transport by a membrane-bound transporter
macromolecule. Finally, plasma proteins are to be mentioned, which may be
viewed as a high-capacity reservoir of drugs in plasma. The significance of
drugs contained within the reservoir is that they are in that state neither
pharmacologically active, nor do they undergo significant clearance
processes.
Biotransformation-based Pharmacokinetic Interactions

A number of prominent drug products have been withdrawn in recent years
because of severe drug-drug interactions and despite preclinical safety

300 Marroum et al.
assessment. Mibefradil, a novel calcium antagonist, for example, was

approved in Switzerland in 1996 and was also launched in the U.S. in 1997
as well as in several other European countries. Shortly following its launch
as an antihypertensive and antianginal agent, reports about serious
pharmacokinetic and pharmacodynamic interactions with other drugs
frequently administered to patients with cardiovascular diseases were noted.
These interacting drugs are to a great extent metabolized by Cytochrome
P450 (CYP450)-dependent microsomal enzymes, including widely prescribed
drugs like quinidine, digoxin, cyclosporin A, terfenadine, and metoprolol. In
addition, reports on severe rhabdomyolysis in patients on mibefradil who
were simultaneously receiving lovastatin or simvastatin were issued.
Mibefradil was reported to mainly inhibit CYP2D6 and 3A4 isoenzymes. In
1998 the drug was withdrawn from the market due to the information
gathered about the severity of drug-drug interactions in patients receiving
mibefradil and other medications [3]. Another example of clinically
important interactions between CYP3A4 inhibitors and drugs largely
eliminated by oxidative biotransformation is between ketoconazole,
itraconazole, clarithromycin, erythromycin, nefazodone, and ritonavir as
inhibitors, when these are coadministered with terfenadine, astemizole,
cisapride, or pimozide.
In that case, Torsades de pointes, a life-threatening ventricular
arrhythmia associated with QT prolongation has been shown to occur as a
consequence of decreased clearance of the arrhythmia-causing parent
compound or metabolite [4]. Finally, a drug-drug interaction between
sorivudine, an antiviral drug, and 5-fluorouracil, an anticancer drug, caused
one of the most serious cases of toxicity ever seen in Japan. The interaction
is based on the irreversible inhibition (mechanism-based inhibition) of
dihydropyrimidine dehydrogenase, a rate limiting enzyme in the metabolism
of 5-fluorouracil by a metabolite of sorivudine, which is formed by gut flora
[5]. On the basis of these case reports on drug-drug interactions due to
decreased metabolic clearance of the active compound and the clinical
experience, several recommendations have been made for the regulatory
assessment of new active substances with respect to drug-drug interactions.
These include the requirement for a detailed understanding about the
mechanism of biotransformation of the parent compound and its
metabolites primarily by in vitro studies with human liver enzymes in which
the potential for metabolic interactions with other drugs is outlined. This
first screen then may serve as a start for identification of drugs that are
commonly used in the target population and that may represent a particular
risk by pharmacoepidemiological studies. Here, particular attention is to be
put on drugs with “a high first-pass metabolism” and “a narrow therapeutic
index.” These may then be studied in interaction studies in the patient
population or in healthy volunteers before their introduction into clinical

practice. Particular attention needs to be put on the interpretation with

respect to the severity of a drug-drug interaction. Here, not only the mean of
the interaction effect, but also the observed and the theoretically
conceivable extreme effects in individual subjects need to be addressed. In
particular, the mibefradil case has shown that for drugs that are expected to
be co-administered in the target population and that may represent a
particular risk, a labelling in the product information indicating the
possibility of an interaction should not be acceptable as a substitute for
performing the appropriate interaction studies before introduction of the
new drug into clinical practice.
Biotransformation-based drug-drug interactions may occur
presystemically, i.e., at the level of the intestine and in the liver
(gastrointestinal and hepatic first-pass effect) and thus may affect the
bioavailability and the clearance of a drug. The intrinsic organ clearance is
defined as:
where V max,i and K m,i are the maximum reaction velocity and
substrateenzyme affinity constant for the ith enzyme. Drug-drug
interactions may affect intrinsic clearance. In the case of competitive
enzyme inhibition, Km is increased, whereas for noncompetitive inhibition,
a decrease in Vmax is noted. Enzyme induction, on the other hand, results in
an increase of Vmax. In particular, for low hepatic extraction drugs (E<0.2),
clearance is primarily dependent upon intrinsic clearance (enzyme activity)
and not liver blood flow. Consequently for these drugs, small changes in
intrinsic clearance, e.g., due to enzyme induction or inhibition, may result
in severe changes of drug clearance. On the other hand, high hepatic-
extraction drugs (E>0.6) have an intrinsic hepatic clearance which exceeds
the hepatic blood flow. Clearance of these drugs is therefore primarily
dependent on liver blood flow and not on intrinsic hepatic clearance. High
ratios of the area under the curves in the presence and absence of an
inhibitor are to be expected when the value of (1+I/Ki) is large, i.e., at high
concentrations of a high affinity inhibitor, and/or when the fraction of the
dose eliminated by a pathway which can be inhibited by the metabolic
inhibitor is large. A particular issue is the relevance of I and Ki values for
the likelihood of an in vivo drug-drug interaction. In the case of reversible
inhibition, a drug-drug interaction (potential for in vivo inhibition) is
considered “highly likely,” if Ki<1 µM and I/Ki>1 [6]. When Ki is between
1 and 50 µM and I/Ki equals 0.1–1, an in vivo interaction is deemed
possible, and when Ki>50 µM and I/Ki<0.1 the potential of an in vivo

302 Marroum et al.
interaction is rather remote. Consequently, if the I/Ki value is larger than

0.3–1, it has been suggested to consider designing the appropriate in vivo
drug interaction studies [7]. The principle has been depicted again
schematically in Fig. 1. It needs to be pointed out though, that the zone of
medium risk is a gray zone and the definition of universal cut-off values is
not uniquely agreed upon by several researchers. Nevertheless, high I/Ki
values for a particular metabolic pathway suggest that the possibility of
occurrence of a drug-drug interaction in vivo because it is likely that the
inhibitor also inhibits other metabolic pathways which have not been
identified yet. For mechanismbased inhibition, Ki values<20 µM for the
inhibitor have “likely” potential for in vivo inhibition, whereas Ki values
in the range of 20 to 100 µM and >100 µM have “possible” and “remote”
potential for causing an in vivo interaction, respectively. The principle has
successfully been applied e.g., for the prediction of the absence of an
interaction between warfarin and tenoxicam, both of which are eliminated
by CYP2C9 [8]. Similarly, an in vivo interaction has been predicted
between warfarin and lornoxicam [8], tolbutamide and sulfaphenazole,
and triazolam and ketoconazole [7]. For the CYP2D6-mediated
dehydration of sparteine and the interaction with the CYP2D6 inhibitor
quinidine, the interaction between the CYP1A2 inhibitor ciprofloxacin
and the CYP1A2 substrate caffeine, and the CYP3A4 substrate
cyclosporin and the CYP3A4 inhibitor erythromycin as well as for the
interaction between the CYP3A4 substrate terfenadine and the CYP3A4
FIGURE 1 Impact of [l]/Ki on the ratio of the AUC of substrate ([S]<Km)in the
presence and absence of a competitive inhibitor. The equation governing the
relationship is: AUCi/AUC=1+[l]/Ki, where AUCi and AUC are the areas under the
substrate concentration-time curve in the presence and absence of the inhibitor,
respectively. The figure was redrawn according to Tucker et al. [14].

inhibitor ketoconazole, the magnitude of the interactions was

underpredicted by factors of approximately 2, 1.5, 1.3, and 7, respectively
[7]. The reasons for this underprediction may include estimation errors for
Ki, the possibility that other elimination pathways may also be reduced by
the inhibitor and the possible accumulation of the inhibitor in the liver.
The latter leads to an underprediction of the inhibitor concentration at the
site of metabolism, which may be the case when carrier-mediated
transport processes promote the uptake of the inhibitor into hepatocytes,
e.g., in the case of ciprofloxacin.
How to predict inhibitory effects of co-administered drugs on hepatic
metabolism of other drugs?
The procedure for predicting the metabolic inhibition by one drug that is
expected to be administered together with the study drug involves several
steps. First, the metabolic pathway of the drug under consideration and
possibly the P450 isozyme(s) most relevant for its degradation should be
identified. This can be done either from metabolic pharmacokinetic drug
interaction databases [9] or it can be determined experimentally e.g., by
human P450 expression systems or by inhibition studies with human liver
microsomes using P450 antibodies or inhibitors specific for each isozyme. A
list of P450 isozymes and their inhibitors is given in Table 1. Secondly,
pharmacokinetic data for the co-administered drug that possibly inhibits the
isozyme responsible for the metabolism of the study drug are assembled and
the maximum concentration of the co-administered inhibitor is estimated.
Thirdly, the Ki of the inhibitor for the metabolism of the study drug is
determined using e.g., human liver microsomes or human P450 expression
systems and the I/Ki ratio is calculated. For more detailed information on in
vitro metabolic methodology, see Chapter 5.
In addition to the selection of a particular in vitro model, particular
probe substrates and inhibitors have to be chosen for the drug-drug
interaction study. Table 1 is a compilation of suitable compounds for
each of the human CYPs. These compounds currently present the most
useful tools to provide in vitro enzyme-kinetic parameters with respect
to the various CYP isoforms [10]. For a variety of reasons, e.g., not
approved as a drug product and/or toxicity in humans, several of the
compounds listed in Table 1 are not suitable for in vivo drug-drug
interaction studies in humans. Therefore, Table 2 contains a list of probe
substrates and inhibitors of CYP isoenzymes which may be used for in
vivo studies in humans. The conduct of in vivo studies is most relevant to
confirm positive outcomes of drug-drug interactions from in vitro
findings and cases are known, in which compounds prove to be potent
inhibitors of CYP isoenzymes in vitro in liver microsomes, yet have no
inhibitory effect on the AUC of various probe substrates in vivo. This
may, for example, be explained by the fact that microsomes are poor

304 Marroum et al.
TABLE 1 In vitro Probe Substrates and Inhibitors for CYPs
1
Can also activate and inhibit CYP3A4.
2
Also inhibits CYP2D6.
3
Also inhibits CYP2C9.
TABLE 2 In vivo Probe Substrates and Inhibitors for CYPs
1
Cannot be administered to healthy volunteers.
2
Also inhibits CYP2D6 at high doses exceeding 150 mg/day.
3
Also inhibits CYP2C9.
4
Also moderately inhibits CYP3A4.
5
Also an inhibitor of 2C19.

performers with respect to phase II metabolic reactions and the scavenging

of potentially inhibitory phase I metabolites is not an issue in whole
functional hepatocytes. An alternative to the use of very specific enzyme
inhibitors in clinical studies is the application of inhibitors with broad
inhibition specificity. Examples include Cimetidine (3A4, 2D6, 1A2, 2C9)
and Ritonavir (3A4, 2D6, 2C9, 2C19). Furthermore, genetic
polymorphisms need to be taken into account. The polymorphic
variability of drug metabolism was empirically recognized before the P450
system was well understood. Slow and rapid acetylators of isoniazid were
recognized in the 1950s. Glucose-6-phosphate dehydrogenase deficiency
leading to hemolytic anemia was appreciated as a genetically based
variation in drug metabolism. In the 1970s, Ziegler and Biggs [15] noted
that African-American patients had significantly higher nortriptyline
levels than did other patients, and these investigators assumed there were
genetic differences [11]. The differences in nortriptyline metabolism are
now believed to result from genetic polymorphisms related to 2D6, 2C9,
and/or 2C19. CYP P450 polymorphisms known today are tabulated in
Table 3. Due to very active research in this field, in particular in the area of
genotyping or phenotyping of individuals with respect to P450 enzymes, it
is expected that this list will continue to grow. Taking the information on
the different metabolism capacities of individuals it may thus be possible
to predict that only those individuals in whom a major metabolic pathway
is inhibited may show profound drug-drug interactions. On the other
hand, the same drug combination may be estimated as having no
interactions, when administered to a subject who is genetically deficient
with respect to the isoenzyme responsible for drug clearance.
In addition to enzyme inhibition, induction processes by some
xenobiotics, both drugs and environmental substances such as cigarette
smoke, may increase the synthesis of P450 proteins. This induction process
may lead to decrease in circulating plasma levels of the parent drug
administered and increase in the concentrations of metabolites produced
and is one of the major underlying mechanisms for time-dependent
pharmacokinetics. For example, co-administration of the potent inducers
rifampin or nevirapine [12, 13], and methadone has led to opiate
withdrawal symptoms. Cytochromes P450 3A4, 1A2, 2C9, 2C19, and 2E1
may all be induced. Important inducers are e.g., carbamazepine,
oxcarbazepine, phenytoin, phenobarbital, rifampin, rifabutin, nevirapine,
troglitazone, dexamethasone, prednisone, St. John’s wort, and primidone
(3A4), tobacco smoke, brussel sprouts, broccoli, cabbage and other
cruciferous vegetables, charbroiled foods, e.g., burned meats (1A2),
rifampin, phenytoin, secobarbital (2C9), rifampin (2C19), alcohol, and
isoniazid [14–17].

306
TABLE 3 CYP P450 Polymorphisms [Cozza, Armstrong, 2001]
Marroum et al.
Numerous examples of documented and clinically relevant drug-drug

interactions exist with respect to enzyme induction. For example, induction
of 3A4 by oxcarbazepine can induce the metabolism of oral contraceptives
rendering them less effective [18]. Plasma concentrations of mirtazapine, a
nonadrenergic and specific serotonergic antidepressant which is mainly
metabolized by CYP 2D6 and CYP 3A4 are decreased by 60% following
enzyme induction by carbamazepine [19]. Rifampicin and rifapentine
induction can decrease plasma concentrations of protease inhibitors or non-
nucleoside reverse transcriptase inhibitors which may lead to viral resistance
(decreased sensitivity to the protease inhibitor or NNRTIs). St. John’s wort
was recently found to decrease mean trough plasma concentrations of
indinavir by 81% (20), cyclosporine A by 43% (21), digoxin AUC by 25%,
and trough concentrations by 33% (22). Interestingly, the effect of St. John’s
wort was only seen following chronic dosing of the hypericum extract and
not after single dose, indicating that the mechanism of action is by induction
of protein expression and not by direct competition with the concomitantly
administered drug.
Transporter-based Pharmacokinetic Interactions

In addition to clearance via phase I or phase II biotransformation
processes, elimination of parent xenobiotics and/or their phase I and phase
II metabolites from the systemic circulation may also be driven by
carriermediated transport. Transporter-related elimination of polar phase
II metabolites has at times been referred to as phase III of drug elimination
[23], although the same terminology has been applied to the metabolism
of phase II metabolites as well, i.e., deconjugation reactions leading to the
reformation of parent compound. Carrier-mediated transport is
particularly important for molecules that would otherwise not be able to
permeate across biological membranes, in particular due to limitations in
their size, charge, or polarity. The liver, the kidneys, and the intestine are
housing the majority of drug transporters responsible for carrier-mediated
drug elimination. In addition to elimination processes, membrane
transporters are having important functions in the organ distribution and
absorption of several drugs. This is schematically depicted in Fig. 2.
According to the human genome project, the estimated number of human
protein-encoding transcripts approximates 30,000 with 26,588 genes
showing strong corroborating evidence [24]. Out of these, 533 are
transporters for inorganic and organic matter. Most transporters are
organized in one out of two superfamilies. These are the “solute carrier
superfamily”, SLC and the “ATP binding cassette,” ABC superfamily.
Currently, 212 genes are family members of the SLC super family
including isoforms, member-like, and antisense sequences as given by the

308 Marroum et al.
FIGURE 2 Schematic tissue distribution and function of some membrane

transporters with respect to disposition and absorption of drugs.
family resources page [25]. A gene is being defined as a DNA segment that
contributes to phenotype/function. In the absence of demonstrated
function a gene is characterized by sequence, transcription, or homology
[26]. Table 4 lists the families of known members of the SLC superfamily
together with a selection of their proposed ligands. Interestingly, only
three out of a total of 32 families are intracellular transporters, the vast

majority is localized at the plasma membrane of cells. Out of these, only a

subset of transporter families is believed to be of relevance with respect to
the transport of drug molecules. These are pointed out in Table 4 and
specific examples of drugs/xenobiotics are given. With respect to the
substrate recognition patterns, the terminology on transporters is
sometimes confusing. For example, members of the solute carrier family
22 include organic cation transporters (OCT1, OCT2, OCT3, OCTN1,
OCTN2) but also anion transporters (OAT1, OAT2, and OAT3). Thus,
members of this family are able to recognize both positively and negatively
charged drug molecules. The second superfamily (ABC family) includes 48
transporter genes. The subfamilies, their names, the number of
transporters in each subfamily, and a selection of proposed ligands are
given in Table 5. The term “ABC” (ATP binding cassette) stems from the
fact, that all members of the ABC superfamily are primarily active
transporters, i.e., the energy for the directed transport of the substrate is
supplied by ATP hydrolysis. “Active transport” is generally characterized
by the requirement for energy, substrate specificity, preferential transport
direction, saturability, and competitive inhibition by cotransported
molecules. The term “facilitated diffusion” on the other hand describes a
process in which the carrier-mediated transport step is not directly coupled
to an energy providing source (most of the SLC transporters). The driving
force is rather provided by an electrochemical gradient across the
membrane (secondary active transport), which is generated, e.g., by the
unequal distribution of positively or negatively charged ions (Na+, H+,
HCO3-, Cl-) across the membrane.
Carrier-mediated absorption, distribution and elimination processes have
in fact been known for a long time. Some examples of substrates for carrier-
mediated absorption are D-glucose, L-dopa, iron (Fe2+), ascorbic acid, small
peptides, penicillins, cephalosporins, angiotensin-converting enzyme
inhibitors, and gabapentin, as has been recognized for some time. With
respect to carrier-mediated distribution, selected uptake into and exclusion
from the blood-brain barrier has been described for e.g., L-dopa, D-glucose,
L-phenylalanine, asimadoline, cyclosporin A, digoxin, colchicine,
vinblastine, and amitriptyline. Furthermore, placental drug passage has
been described to be modified by membrane carrier proteins that are
expressed in the maternal-facing brush-border membrane and the
fetalfacing basal membrane of the syncytiotrophoblast, the polarized
epithelium, and the functional unit of the placenta. Examples include
digoxin [27], valproic acid [28], monoamines (serotonin, dopamine,
amphetamine, imipramine), clonidine, cimetidine, and amiloride as well as
cephaloridine [29]. In addition, various transporters for monocarboxylates
(MCT1, 3, 5, 5, and 7) and dicarboxylate have been found. A summary of
characterized transporters expressed in placenta is given by Ganapathy [30].

310
TABLE 4 Family Members of the SLC Superfamily of Solute Transporters
Marroum et al.
312
TABLE 4 Continued
Marroum et al.
TABLE 5 Family Members of the Human ABC Superfamily of Solute

Transporters
Carrier-mediated excretion in the liver and/or kidney and/or gastrointestinal

tract has been described for p-amino-hippuric acid, penicillins,
cephalosporins, digoxin, doxorubicin, fluvastatin, lovastatin, vincristine,
quaternary ammonium compounds, ciprofloxacin, and indocyanine green.
Relatively new is the knowledge of carrier-mediated anti-absorptive
transporters in the intestine. It has been difficult for some time to
differentiate between intestinal exsorption and metabolism, since both may
reduce the amount entering the portal blood in a dose-dependent manner
leading to low bioavailability at the low dose level and higher bioavailability
at higher dose levels. Differentiation in in vivo studies—on the clinical
level—appears to be possible only by selective inhibition using process-
specific inhibitors [30, 31].
Intestinal exsorptive transporters became evident as soon as highly
specific, potent, and low-dosed drugs were developed. Limited peroral
bioavailability or lack of bioavailability of various newly developed
compounds were indicative of bioavailability-limiting processes. Likewise,
carrier-mediated transport across the membranes of the blood-brain
barrier, the kidney, and the liver were recognized for some time to
contribute to the distribution and clearance of a drug from the systemic
circulation.

314 Marroum et al.
Carrier-mediated absorptive or exsorptive processes are saturable and

inhibitable. In Fig. 3, the relationship between the administered dose and the
bioavailability or fraction dose absorbed for saturable exsorptive and
absorptive processes is demonstrated schematically. Each transporter may
be characterized with respect to Km and Vmax, i.e., regarding the substrate
concentration at which half-maximal transport velocity and maximum
transport rate are observed. The overall contribution of the respective
transport processes in the absorption of the drug determines the relevance of
the saturable mechanism. High passive permeabilities significantly reduce
the relevance of carrier-mediated inside- or outside-directed transport
processes, although the affinities of the substrate to the respective
transporters may be high. Similarly to drug metabolism, in principle, two
different types of drug-drug interaction mechanisms are feasible with
respect to compounds, which are substrates for transporters:
a. Inhibition (reduction) or enhancement of drug transport through

competitive/noncompetitive inhibition of binding, or transport or
increase of transport through interaction with the transporter, and
FIGURE 3 Potential for drug-drug and drug-food interactions at the presystemic

level: First-pass metabolic or secretory processes on the one hand, or absorption
via an active process on the other hand may cause nonlinearities of the
bioavailability vs. dose relationship. The relative contribution of the saturable
process is reduced upon increasing dose. Inhibition of the respective metabolic or
transport process leads to a partial or complete disappearance of nonlinearity.

b. Alteration of transporter expression, i.e., changes of the number

of protein molecules available for the transport of drugs
(induction or reduced expression).
Most relevant in this respect appear to be transport inhibition and induction

of transporter expression. Transport inhibition in the intestine, for example,
leads to a decrease of bioavailability, when an inside-directed transport
process is reduced or inhibited. Figure 4 illustrates the changes in
transepithelial drug flux based on concomitant absorption via a
carriermediated process, passive diffusion, and exsorption (secretion), when
competitive inhibition occurs affecting active, yet not passive transport
processes. Inhibition of secretory transporters leads to enhanced drug flux
across the membrane in the absorptive direction (Fig. 5). On the other hand,
in the case of induction of secretory transporters (Fig. 6), transepithelial
apical to basolateral fluxes decrease as soon as the expression of the
FIGURE 4 Inhibition of transport as interaction mechanism: The relevance of the

active inside- or outside-directed transport process depends on the ratio between
active and passive processes. When the passive, nonsaturable, and noninhibitable
process is dominating, the interaction potential and the potential for nonlinearity are
reduced.

316 Marroum et al.
FIGURE 5 General mechanisms for drug-drug and drug-food interactions in the

intestine A: Competitive or non-competitive inhibition. Intestinal secretion
(exsorption) was chosen as an example for one isolated mechanism of usually
many. Like in biotransformation, inside- and outside- directed transport processes
may be saturated upon high substrate levels and be inhibited by other compounds
with affinity to the respective relevant binding sites. In the case of intestinal
secretion, inhibition of the process leads to a higher intestinal permeability and an
increase of absorbed fractions.
secretory carrier is increased. For induction of transporters responsible for

an absorptive carrier-mediated process, increased apical to basolateral
fluxes are to be expected, i.e., bioavailability should be enhanced.
In general, two different scenarios need to be considered with respect to
DDIs at the transport or metabolism levels, respectively:
1. A drug affects the kinetics of a co-administered compound.

2. A drug is affected by a co-administered compound.
Examples illustrating the relevance of the abovementioned mechanisms

with respect to DDIs are beginning to emerge. For example, co-
administration of substrates of the efflux transporter P-glycoprotein, the
product of the multidrug resistance gene (MDR1 in humans), have been

FIGURE 6 General mechanisms for drug-drug and food-drug interactions in the

intestine B: Induction. Like in biotransformation, induction of intestinal secretion
leads to a decreased intestinal permeability via outside-directed transport and a
reduction of the absorbed fraction. Due to induction of exsorption, the relative
influence of secretion inhibitors may be higher in the induced state.
well described to result in pharmacokinetic drug-drug interactions.

Examples include the interaction of the ß-adrenoceptor antagonist
talinolol when administered together with verapamil [31, 32] and
erythromycin [33]. From some of these interaction studies it could be seen
that the bioavailability of talinolol (increase in rate and extent of
absorption) increased when administered together with the co-medication.
Also it has been demonstrated by a variety of in vitro, in situ, and in vivo

318 Marroum et al.
techniques, that intestinal secretion of talinolol is saturable and

inhibitable by several P-gp modifiers. A much less pronounced effect of the
co-medication was observed with respect to changes in talinolol
elimination half-life in clinical studies. Nevertheless, preclinical studies
have shown [34] that the distribution of talinolol can be significantly
modified, when another P-gp substrate or inhibitor is co-administered.
Interestingly, also food and food components have recently been described
to interact with carriers [35, 36] thus serving as another possibility of
explaining peculiar food-drug interactions. Digoxin, another P-gp
substrate, is likewise eliminated mainly via excretion of the unchanged
moiety. This means, that metabolic drug-drug interactions as the major
underlying mechanism of the DDI can be virtually excluded. Digoxin has
been described to interact with several P-gp substrates/inhibitors, e.g.,
talinolol [37, 38], propafenone [39], verapamil [40, 41], quinidine [42],
itraconazole [43], ketoconazole [44], clarithromycin [45], rifampin [46],
valspodar [47], and atorvastatin [48]. In addition, also for herbal extracts
such as extracts of St. John’s wort (Hypericum perforatum), which are
frequently used as over-the-counter medication, a pharmacokinetic
interaction with digoxin has been reported [49]. Interactions of St. John’s
wort have been reported also in the case of cyclosporine and indinavir,
however for the latter two, a contribution of cytochrome P4503A4 to the
overall extent of the interaction must be taken into account. Since virtually
all of the abovementioned drugs show affinity to P-glycoprotein,
competition for the binding site or modulation of the function of the
multidrug resistance gene product has been made responsible for the
observed drug-drug interactions. Since P-glycoprotein is widely
distributed in absorbing and eliminating epithelia, e.g., small and large
intestine, liver and kidneys—among other noneliminating tissues—the
increase in digoxin plasma AUC has been attributed to be the result of a
decrease in digoxin renal tubular secretion, which is suggested to be
mediated by P-glycoprotein, and an increase in digoxin absorption in the
GI-tract in the presence of the co-administered drug. Most probably, also
altered distribution phenomena have to be taken into account, since it has
been shown e.g., in studies with mice, that co-administration of quinidine
may increase digoxin brain concentrations in wild-type mice, whereas no
increase was reported for the P-glycoprotein deficient mdrla(-/-) knockout
mice [50]. Furthermore, a clinically significant interaction at the blood-
brain barrier has been described for quinidine, increasing loperamide-
induced central effects in humans [51]. Similarly, it is well known that P-
glycoprotein is expressed on the brush-border membrane (maternal side)
of human placental trophoblast cells and is considered to regulate the
transfer of several substances including vinblastine, vincristine, and
digoxin from mother to fetus, and to protect the fetus from toxic

substances [52]. Consequently it may be hypothesized, that co-

administration of a P-glycoprotein modulator together with a P-
glycoprotein substrate may severely affect the drug concentrations in the
fetus. The AUC increase of digoxin upon comedication is generally
dependent on the type, dosing regimen, and dose of the co-administered
drug. In the case of verapamil, it has been found that digoxin plasma
concentrations rose by 60 to 90% [41], whereas with the high affinity
modulator valspodar upon multiple dosing, an increase in digoxin AUC of
more than 200% has been reported [47]. Since other digitalis glycosides
such as digitoxin, α-methyldigoxin, and ß-acetyldigoxin are also
substrates of P-glycoprotein [53], it may be hypothesized that similar
drugdrug interactions exist for these drugs. Most of the abovementioned
drugs are lipophilic and carry at physiological pH—at least to a partial
extent—a positive charge. This renders such molecules susceptible to P-
glycoproteinmediated transport. On the other hand, transporter-based
drug-drug interactions have also been described for a number of organic
anions. For example, the loop-diuretic furosemide is subject to polarized
transport across renal and intestinal epithelia [54]. The secretion of
furosemide can be inhibited with indomethacin. Indomethacin has long
been known to inhibit renal clearance of many anionic xenobiotics [55,
56], however, this has not yet been attributed to a single transporter.
Instead, the involvement of several transporters is discussed, such as
kidney organic anion transporters (OAT), for which p-aminohippurate
serves as endogeneous ligand [57] and the Multidrug Resistance-
Associated Protein (MRP) transporters.
Many drug-drug interactions arise from concurrent administration of
drugs which are both substrates and inducers of CYP3A4 and MDR1
expression. Long-term therapy with drugs that induce CYP3A4 and MDR1,
for example, increase the systemic clearance of some antileukemic agents,
and such therapy has been shown to exert negative effects on survival while
increasing cancer relapse [58]. Recent studies have shown, that the steroid
and xenobiotic receptor (SXR), a member of the nuclear hormone receptor
subfamily which is expressed in the liver and also in the intestine, has a
central role in regulating CYP3A4, CYP2C8, and P-glycoprotein
transcription via a coordinated mechanism [59, 60]. Thus it is
mechanistically understandable, why some Pharmaceuticals such as
rifampicin or St. John’s wort, induce both the formation of metabolic
enzymes as well as the expression of P-glycoprotein. Steroid and xenobiotic
receptor thus shows an ability to coordinately regulate multiple xenobiotic
clearance pathways and could be regarded as a “steroid and xenobiotic
sensor” with a central role of balancing xenobiotic input and elimination as
a function of their concentration in the body. Steroid and xenobiotic
receptor is activated by a pharmacopoeia of drugs including antibiotics,

320 Marroum et al.
HMG-CoA reductase inhibitors, antiseizure medications, steroids such as

glucocorticoids, environmental contaminants such as organochlorine
pesticides and polychlorinated biphenyls, and herbal supplements such as
St. John’s wort. It needs to be investigated, whether screening for SXR
affinity is an appropriate measure to distinguish between SXR transparent
drugs and potential enzyme inducers. An example on how the safety of a
drug can be improved by avoiding SXR affinity is given by the structurally
closely related chemotherapeutic agents paclitaxel (Taxol) and docetaxel
(Taxotere). Whereas paclitaxel activates SXR and thereby induces its own
clearance in a time-dependent manner, docetaxel does not activate SXR or
induce drug clearance [60].
In recent years, evidence begins to emerge that variability in
pharmacokinetics and drug response may also be in part due to
polymorphic variability of drug transporters. For example, for the MDR1
gene (ABCB1), 15 different polymorphisms have been reported, 12 of
which did not alter the protein sequence [61]. The mutant C3435T at exon
26 was associated with a lower level of MDR1 expression in enterocyte
preparations of the duodenum which was determined by Western blot
analysis (P=0.056; n=21). It was also suggested that this exon 26 single
nucleotide polymorphism (SNP) correlated with the pharmacokinetics of
digoxin, whereby the steady-state Cmax values of digoxin were 38% higher
in volunteers carrying the T/T genotype as compared to the C/C genotype.
Another study in 114 healthy volunteers in a Japanese population [62]
also performed MDR1 genotyping at exon 26. For the wild-type allele (C/
C) 35.1% of the population were found homozygous, 52.6% were
heterozygotes (C/T), and 12.3% were homozygous for the mutant allele
(T/T). Interestingly, serum digoxin concentrations (AUC0–24h following
single dosing) were found to be lower in subjects harboring the mutant T-
allele, i.e., C/T and T/T. A satisfactory explanation for the discrepancies
between these apparently contradictory findings has yet to be given. In the
case of fexofenadine, the C/C mutant resulted in significantly higher
plasma concentration-time profiles after peroral administration indicating
lower P-gp activity or expression levels in this genotype [63]. An effect of
gender or age on the genotype distribution could not be found. However, a
sitedirected Ser893 mutation instead of Ala893 in the P-glycoprotein
sequence caused by two synonymous SNPs (C1236T in exon 12 and
C3435T in exon 26) and a nonsynonymous SNP (G2677T) in exon 21
were found to be linked and occurred in 62% of the European Americans
and only in 13% of African Americans, indicating a possible ethnic
component in the population distribution. This mutation was significantly
correlated with a higher in vitro activity of P-glycoprotein and lower in
vivo plasma concentrations of the P-gp substrate fexofenadine in healthy
subjects [63]. Ethnic differences in MDR1 polymorphisms were also

confirmed by a recent study, in which the C3435T mutation was profiled

in 1286 research participants from ten different ethnic groups in which
significantly higher frequencies of the C/C genotype were found in West
Africans and African Americans than in Whites and Japanese populations
[64]. An interesting study on the implications of polymorphisms of MDR1
as well as CYP3A4, CYP3A5, CYP2D6, and CYP 2C19 on the
pharmacokinetics and dynamics of nelfinavir or efavirenz in HIV-infected
patients suggested that plasma concentrations of both antiretroviral drugs
decreased in the order of. C/C>C/T>T/T allelic variations in the MDR1
gene [65]. The pharmacodynamic effect of the antiretroviral therapy
quantified as an increase in the CD4-cell count was greatest in the T/T
genotype, followed by the C/T and C/C genotypes. The finding that P-gp
expression in peripheral blood mononuclear cells was lowest in the T/T
genotype suggests that the MDR1 polymorphism has significant
implications with respect to the admittance of antiretroviral drugs to
restricted compartments in vivo. Other hereditary polymorphisms in ABC
drug transporters (MRP1, MRP2) are the subject of current investigations
[66, 67].
With respect to drug-drug interactions, the consequences of genetic
polymorphisms still have to be determined. It may be hypothesized that
polymorphisms in transporters which are involved in the ADME cascade
of substrates will most likely contribute to the between-subject
variability of a drug-drug interaction. The magnitude of the variability
will depend on the level of expression of functional transporter protein,
the affinity of both drugs to the transporter and the concentration-time
profiles of the drugs in the respective organs in which the transporter is
expressed. Initial data on the magnitude of transporter induction e.g., by
rifampicin also suggest that the magnitude of transporter induction is
dependent on the genotype, as has been shown for the MDR1 gene
product P-gp [61].
In order to screen for potential DDIs, it may be helpful to study the
compound of interest together with certain model compounds. In the
literature several compounds appeared, which were found to exhibit affinity
to P-glycoprotein and were studied mainly because a DDI was highly
probable. This group of compounds is characterized by a certain
intermediate hydrophilicity/lipophilicity and intermediate passive
permeability. It includes digoxin, fexofenadine, and talinolol (Fig: 7). A
highly lipophilic, poorly metabolized, and well-diffusing compound—
although a P-gp substrate—may readily pass across membranes and be
almost completely absorbed.
With a restriction for talinolol, which is marketed in Europe only, the
compounds are well accessible. Moreover, all three have become
commercially available as tritium-labeled compound permitting the rapid

322 Marroum et al.
FIGURE 7 Chemical structure of commonly used P-gp substances.
assay of samples from experimental and mechanistic transport studies (in

vitro, in situ, in vivo in animals).
Regarding their kinetic behaviour in man, the three drugs have further
similarities: They are metabolized to a negligible extent only, i.e., have a
high unchanged metabolic clearance, while metabolic substrate loss does
not play any significant role. The high therapeutic range observed with the
ß-adrenoceptor antagonist talinolol and with fexofenadine represents a
considerable advantage over digoxin. Selection of an appropriate model
compound may be based on the expected side-effects, on the availability of
the respective compound and legal considerations.
Digoxin: Potentially because of a favourable passive/active transport
ratio, and its traditional availability as a radioactively labeled compound,
and in spite of a fairly narrow therapeutic range digoxin has been used as a
P-gp model substrate to be influenced by concomitantly administered drugs
[50, 68–71]. Its worldwide availability may be an additional reason for its
selection.
Talinolol: The use of talinolol as model compound for transport-related
processes in, e.g., drug-drug or drug-food interaction studies as previously

proposed [36] appears reasonable because of its mainly unchanged renal

and biliary clearance, the low protein binding (approximately 25%), and
the sensitivity of its kinetics for changes in P-glycoprotein expression, but
also to transporter function (inhibition by P-gp modulators). Only to a small
extent these advantages are neutralized by a potential for affinities to other
transporters: 3H-Tetraethylammonium uptake studies in LLC-PK1 cells
revealed an inhibiting effect of talinolol on TEA uptake, which indicates an
additional interaction with the OCT [72]. Furthermore, there is evidence
from in vivo studies with MRP2 deficient rats that MRP2 also contributes to
talinolol disposition to some extent.
It may be considered to use the distomer instead of the racemate, since the
eudismic ratio for talinolol is approximately 40 and no significant
Pglycoprotein affinity difference was detected for the enantiomers.
Fexofenadine: Fexofenadine, a nonsedating antihistamine and
metabolite of terfenadine does not—like talinolol—undergo significant
metabolic biotransformation. Employing different cell lines, evidence was
found that uptake and efflux transporters are involved in fexofenadine
absorption and disposition [73, 74]. Among various transport systems
investigated, the human organic anion transporting polypeptide (OATP)
and rat organic anion transporting polypeptides 1 and 2 (Oatpl and Oatp2)
were identified to mediate [14C]-fexofenadine cellular uptake, while P-gp
was identified as fexofenadine efflux transporter, using the LLC-PK1 cell, the
polarized epithelial cell line lacking P-gp, and the P-gp overexpressing
derivative cell line L-MDR1. Studies in P-gp knock-out mice confirmed the
relevance of this transporter for fexofenadine disposition in a similar way as
demonstrated for talinolol, for which a high relevance of P-gp for
absorption and disposition was detected [75].
Interactions as a Result of Alterations in Plasma Protein Binding

Competition for protein binding sites is likely when two drugs are highly
bound to plasma proteins. The displacement of a drug from its binding site
at the protein is frequently followed by an increase in its unbound drug
concentration. Since it is the unbound drug that is pharmacologically active,
this increase in “free” drug tends to increase the pharmacodynamic effect of
the displaced drug. The conditions favoring displacement have been
outlined previously [76]. In addition to the type and concentration of the
respective binding protein (600 uM for albumin and 9–23 uM for α1-acid
glycoprotein) the plasma concentrations of the drug and the displacer and
their affinities to the binding sites are of relevance. Displacement of drugs
that bind to α1-acid glycoprotein is more likely to occur as a result of the
lower blood concentrations of this protein as compared to albumin. An
initial increase in the unbound plasma concentration of a low extraction

324 Marroum et al.
drug (restrictively cleared drug) may however be readily compensated by an

increase in its clearance, and an additional buffering effect by an increase in
its volume of distribution. Thus, although total drug plasma concentrations
may be diminished in an interaction situation, the unbound concentrations
of the drug may remain constant and no dosage adjustment needs to be
made. An example is the displacement of phenytoin by valproic acid.
Coadministration of valproic acid to phenytoin has been reported to
decrease total steady-state plasma phenytoin concentrations in a dose-
dependent manner [77]. In accordance with the theory, unbound
concentrations of phenytoin remained constant in that study. On the other
hand, the theory of plasma protein binding displacement interactions being
the common cause of clinically significant interactions has been questioned
[78]. In the case of valproic acid and phenytoin, additional mechanisms are
likely to be the major ones responsible for the exaggerated effect observed
clinically [19]. In addition to the displacement from plasma protein binding
sites, going along with an increased distribution of the drug throughout the
rest of the body and concomitant enhancement of the systemic clearance of
total drug, an inhibition of phenytoin metabolism by valproic acid and
thereby an increase in the concentration of free drug in the serum has been
described [80]. Likewise additional mechanisms are likely to be involved in
the causes of drug-drug interactions with clinically observed exaggerated
effects, e.g., the interaction of warfarin with phenylbutazone leading to
marked increases in prothrombin times and the interaction of
sulphonamides with tolbutamide resulting in a sustained increase in
hypoglycaemic effect, as well as the toxic interaction between acetazolamide
and salicylate [81]. In all cases, a reduction of the clearance of free drug has
been made responsible for the accumulation of the displaced drug, thus
making the hypothesis of a drug-drug interaction purely driven by plasma
protein displacement unlikely. For high clearance drugs (unrestrictively
cleared, flow-limited) administered intravenously, increased free
concentrations following displacement will not be adequately compensated
by increased clearance, as both free and bound drugs are already available
for elimination by the clearing organ and clearance will be most sensitive to
changes in organ blood flow rate. Thus the increased free-drug
concentrations will possibly result in an enhanced response. Examples for
drugs, where protein-binding displacement may be clinically significant
include lidocaine, alfenanil, buprenorphine, fentanyl, hydralazine,
midazolam, and verapamil [82]. For nonrestrictively cleared drugs (hepatic
clearance) which are given perorally, the increase in the free fraction may
cause a slight increase in hepatic extraction and a decrease in bioavailability,
which will lead to a reduction in steady-state concentrations (Css). The
combined effect of an increase in fu and a decrease in Css, however, means
that unbound steady-state concentrations of the drug being displaced will be

largely unaltered compared with the predisplacement value. There are very
few perorally administered drugs that exhibit the properties of extensive
plasma protein binding and high hepatic first-pass extraction, for example
propranolol, imipramine, and desipramine. Those, however, tend to have a
relatively wide therapeutic margin.
Physicochemical Interactions and Interactions based on

Changes in Local pH and lonization State of Molecules,
Respectively
A few drugs have structures that readily form chelate complexes with
divalent or trivalent cations such as aluminium, magnesium, iron, or
calcium. The complexed drugs are not absorbed across the intestine and
hence their plasma concentrations may be subtherapeutic. Examples include
quinolone antibiotics (e.g., ciprofloxacin) and tetracyclines which are
markedly less absorbed when administered together with magnesium-
aluminium antacids. Other cations, such as calcium, iron, and probably
zinc, appear to interact in a similar manner. Cholestyramine is a basic anion-
exchange resin used in the treatment of hypercholesterolemia. The
hydrophilic but water insoluble powder is not absorbed in the GI tract,
however, it can adsorb bile acids and a number of drugs (e.g., digitalis
glycosides, coumarin, diuretics, quinidine, thyroxine, propranolol, and
some antibiotics). As a safety precaution it has been recommended to
discontinue resin administration for short-term courses of antibiotics,
corticosteroids, pre- and postoperative medications, rather than risking the
possibility of the action of the drugs being diminished or abolished by the
interaction with the resin. Malabsorption of lipophilic drugs has also been
observed when the drugs were administered together with nondigestible
oils. Here, it is likely, that the drugs will dissolve in the oil and thus may not
be available for absorption [83, 84]. In some instances, nondigestible oils
have been used for enhancing the intestinal elimination of toxicants [85,
86]. Other studies have shown though, that upon proper spacing of the
intake of nonabsorbable fat replacements and lipophilic drugs, an
interaction can be avoided [87, 88]. Activated charcoal is another drug with
several potential interactions based on surface adsorption. This is due to its
large surface area of approximately 1000 m2g-1, which however varies from
one charcoal preparation to another. Some of the documented interactions
include anticonvulsants and oral emetics as well as oral antidotes for
acetaminophen poisoning such as methionine [89]. Drug-drug interactions
based on changes in the ionization state of molecules are of particular
relevance for processes and compartments, in which significant changes in
the local pH occur. Such compartments are the kidneys and the stomach.
The pH in the stomach may vary considerably, also as a function of

326 Marroum et al.
co-administered drugs. For example, the median pH in a control group of

gynecologic out-patients increased from 2.2 to 5.7 following treatment with
400 mg of cimetidine [90]. As a consequence, the dissolution and absorption
of basic drugs with low water solubility, such as ketoconazole, is diminished
in cases of lowered gastric acidity [91]. Similar observations have been made
for itraconazole.
Changes in urinary pH may alter the tubular reabsorption of drugs with
pka values in the physiological range. Thus weak acids such as salicylic acid,
barbiturates, and sulfonamides show higher renal clearance at alkaline urine
pH. On the other hand, weak bases, such as amphetamine, antihistamines,
imipramine, and meperidine are preferentially cleared at acidic urinary pH
values (approximately 5).
Drug-drug Interactions based on Pharmacodynamic-

Pharmacokinetic and Pharmacodynamic Mechanisms
Pharmacodynamic-pharmacokinetic drug-drug interactions originate from
situations, where a pharmacological effect of a particular drug can modify
the pharmacokinetics of a second drug. For example, a compound which
affects the gastrointestinal motility may influence the rate and extent of
absorption of another co-administered drug by altering gastric emptying
times and passage times across the small intestine. Thus the absorption of
paracetamol can be delayed with concurrent administration of
propantheline, a muscarinic receptor antagonist and with opiate-type
analgesics. Metoclopramide and other prokinetic agents however, increase
motility and transit of material in the gastrointestinal tract. The question as
to whether the extent of drug absorption of a particular compound is
modified by a prokinetic agent is frequently dependent on the intestinal
permeability of the drug. For compounds with high permeability, the extent
of drug absorption remains unchanged, since the residence times in the
absorbing segments are more than sufficient to ensure complete absorption.
Thus, even an increase in the gastrointestinal transit times will manifest in a
change in rate but not extent of drug absorption. Another example for a
pharmacodynamic-pharmacokinetic interaction is the interaction between
compounds which modify the blood flow through the major clearing organs
and high-clearance drugs. Propranolol, for example, by reduction of the
cardiac output, diminishes the liver blood flow and reduces its own
clearance and the clearance of lidocaine and bupivacaine [92, 93]. Similar
interactions due to modification of blood flow in target tissues have been
observed with anaesthetic agents. For example, volatile anaesthetics have
been shown to delay the intramuscular absorption of ketamine in addition
to diminishing the volume of distribution and clearance of a number of
high-clearance compounds [94].

REGULATORY ASPECTS OF DRUG-DRUG INTERACTIONS
FDA Guidance on in vivo Metabolic Drug Interactions

Studies
In November of 1999, the FDA issued a guidance on the study design, data
analysis, and recommendations for dosing and labeling of in vivo metabolic
drug interaction studies [95]. The basic concepts that are behind the
recommendations in this guidance are as follows:
1. An understanding of the metabolic fate of a drug and the

contribution of metabolism to the overall elimination of the
drug is essential in the assessment of its safety and efficacy
profile.
2. It is important to elucidate whether the investigational drug
affects the metabolism of currently marketed drugs and
conversely whether the metabolism of the investigational drug is
also affected by currently available drugs.
3. Sometimes even though a drug might not be metabolized, it still
can be a potent inhibitor of a certain metabolic pathway. Thus it
is important to elucidate its effect on the metabolism of currently
marketed drugs metabolized by the inhibited enzymes.
4. The clinical importance of a drug interaction sometimes depends
on the genetic polymorphism (whether a patient is considered a
slow or fast metabolizer) of the individual. Moreover, other
covariates such as age, race, and gender can be of prime
importance in the clinical outcome of the interaction.

Dosing Regimens. One of the major considerations in designing a drug-drug
interaction study is whether to dose the substrate (S) or the interacting drug
(I) as single dose or chronically (multiple dose).
The selection of the dosing regimens will depend on
a. Whether the S or I is dosed acutely or chronically in the clinical

setting
b. Safety considerations including whether the drugs are considered
narrow therapeutic index or not
c. The pharmacokinetic and pharmacodynamic characteristics of
the S and I
d. The need to assess induction or inhibition.

328 Marroum et al.
A recent survey of all approved new molecular entities, approved between

1992–1997, showed that the preferred dosing regimen was to dose both I
and S to steady state (47% of all studies) while in 30% of the cases one of
the drugs was dosed to steady state [96]. The use of such designs is a
reflection of the clinical use of these drugs and the fact that for inducers and
some inihibtors it might take several days to see the full extent of the
interaction. As an illustration to this point, an interaction study between
alfentanyl and erythromycin did not show any interaction on the clearance
of alfentanyl. However, after a seven-day course of 500 mg erythromycin
twice daily, there was a 25% decrease in alfentanyl clearance and a 60%
increase in the alfentanyl half-life [97]. Another complicating factor in the
ability to extrapolate the single dose findings to steady-state situations is the
potential for certain inhibitors to also act as inducers when given on a long-
term basis. One such drug is the protease inhibitor ritonavir.
On the other hand, the vast majority of absorption-based drug
interaction studies with drugs such as antacids or drugs that affect gastric
motility use a single single-dose study design since with this design one can
determine whether the bioavailability of the S is affected.
Study Population
The vast majority of drug-drug interaction studies employ healthy
volunteers as the study population since it is assumed that the findings
obtained from such a population can easily be extrapolated to the patient
population for which the drug is intended. However, in certain instances
where safety considerations precludes the use of healthy volunteers, or in
situations where the pharmacodynamic endpoints to be measured in the
study cannot be easily extrapolated to the patient population, one is forced
to recruit from the general patient population. In either case, performance of
genotype or phenotype determinations to identify genetically determined
metabolic polymorphisms is often important in evaluating enzymes such as
CYP2D6 or CYP2C19.
Statistical Design Considerations

The number of subjects to be enrolled in the study depends on the
magnitude of the effect to be detected that is considered to be clinically
relevant, the inter and intrasubject variability in the PK measurements and
any other factors that might affect the outcome of the study.
The most common statistical design for pharmacokinetic drug
interactions is the crossover design accounting for half of all the studies
submitted to the Agency from 1987 to 1997. More recently an increased
reliance on a fixed sequence design (where a subject receives a drug for a

fixed period and the second drug is introduced at a certain time in the dosing
period). Such a design is considered to be a variation of the crossover design.
A parallel design is most useful in situations where one of the studied drugs
or its metabolites have a long half-life.
According to the FDA guidance, the results of the drug-drug interaction
studies should be reported as 90% confidence intervals about the geometric
mean ratio of the observed PK measure with and without the interacting
drug. Confidence intervals will provide an estimate of the distribution of the
observed systemic exposure with and without the interacting drug and thus
conveying a probability of the magnitude of the interaction. On the other
hand, tests of significance are not appropriate for such studies due to the
fact that clinically insignificant exposure differences can achieve statistical
significance without having to recommend dosing adjustments or
contraindications.
Moreover, the FDA guidance recommends that in a drug-drug interaction
study, the sponsor of the investigational drug should be able to provide
specific dosing recommendations based on what is known about the PK/PD
relationship or the dose-response relationship. Unfortunately such
information is not always available especially for drugs that are already on
the market.
If the sponsor intends to make a specific claim in the package insert that
no drug interaction is present, the sponsor should be able to recommend
specific “no effect boundaries” or clinical equivalence intervals defined as
the interval within which the change in a systemic exposure measure is
considered to be clinically not relevant.
The guidance recommends three approaches in defining these no effect
boundaries:
Approach 1:
The no effect boundaries are based on population average dose-response or
exposure-response relationships and any other available information for the
drug under study. If the 90% confidence interval for the systemic exposure
measure falls within the no effect boundary, then it may be concluded that
no clinically significant drug-drug interaction is present.
Approach 2:
The no effect boundary may also be based on the concept that a drug-drug
interaction study addresses the question of switchability between the
substrate given alone and in combination with an interacting drug. In this
case, a sponsor may wish to use an individual equivalence criterion to allow
for scaling of the no effect boundary.

330 Marroum et al.
Approach 3:
In the absence of no effect boundaries as defined in Approach 1 or 2, a
sponsor may use a default no effect boundary of 80–125% for both the
investigational drug and the approved drugs used in the study. When the
90% confidence intervals for systemic exposure fall entirely within the
equivalence range, the Agency in most cases will conclude that clinically
significant interaction is present.
It is to note that Approach 3 does not necessary imply that the
sponsor needs to always power the study in a way that the 90%
confidence interval for the ratio of pharmacokinetic measurements falls
entirely within the no effect boundary resulting in an increased number
of subjects for each study.
Choice of Substrate and Interacting Drugs

Substrates for an Investigational Drug. If the investigational drug is an
inhibitor of a specific enzyme system, the substrate to be selected as the
interacting drug should be one whose pharmacokinetics is markedly altered
by the inhibitor. The guidance includes several examples of substrates such
as midazolam, buspirone, felodipine, simvastatin or lovastatin for CYP3A4,
theophylline for CYP1A2, S-warfarin for CYP2C9, and desipramine for
CYP2D6. If the initial study was found to be positive, further studies of
other substrates might be recommended based on the likelihood of
coadministration. If the initial study was found to be negative with the most
sensitive substrate, then it is safe to assume that the less sensitive substrates
will also not be affected.
Investigational Drug as a Substrate. The testing of the investigational
drug as a substrate will depend on the results of the in vitro metabolic
studies identifying the enzyme systems that metabolize the drug. If for
example the investigational drug is shown to be metabolized by CYP3A4 to
a great extent, the choice of inhibitor and inducer could be ketoconazole or
rifampin since both of these drugs are known for their substantial effect on
this pathway. If the results of such a study are deemed negative, then the
absence of an interaction for this metabolic pathway could be claimed by
the sponsor. However, if the study found a clinically significant interaction
for this metabolic pathway, and the sponsor would like to claim a lack of
interaction with a less potent inhibitor/inducer then more studies would be
recommended to substantiate the specific claims with regard to the less
potent interactants.

Route of Administration
In general, it is recommended that both the substrate and interacting drug be
administered in the same way these drugs are used (or going to be used
clinically). However, if multiple routes of administration are possible, it
might be necessary in some cases to investigate the possibility of drug
interactions with the different routes of administration. This is particularly
true for drugs that undergo gut wall metabolism whereby the amount of
metabolism will differ between the oral and intravenous routes. Therefore it
is thought that the differences in exposure that result from a drug
interaction will be different depending on the route of administration
(viagra interaction with erythromycin), which will consequently result in
different dosing adjustment recommendations, then in such cases one is
better off obtaining the true magnitude of interaction for the different routes
of administration.
Dose Selection
Unless there are overriding safety concerns it is recommended to use the
highest possible dose for both the substrate and the interacting drug and the
shortest dosing interval. This will maximize the probability of finding an
interaction and will also shed light on the possible maximal magnitude of
the interaction and the worst case scenario in the change in exposure that
will result in a clinically significant interaction such as dosing adjustment or
even a recommendation to contraindicate the co-administration of the two
drugs.
Labeling
The FDA guidance recommends the inclusion of both positive and relevant
negative findings of the results of the in vivo drug interaction studies in the
“Clinical Pharmacology” section under “drug-drug interactions.” If the
results of the study indicate a potentially clinically significant interaction or
the lack of an important interaction that might have been expected, in
addition to mentioning it in the “Clinical Pharmacology” section, a more
detailed description of the study and its results should be included in the
“Precautions” section of the label with advice on how to adjust the dosage
in the “Warnings/Precautions,” “Dosage and Administration,” and
“Contraindications” sections of the label. The FDA guidance allows the
extrapolation of the results of a drug-drug interaction study with a certain
substrate or inhibitor to other substrates or inhibitors/inducers not
specifically tested thus allowing for a class label based on the results of the
study with a drug that is considered a prototype. For example, if an

332 Marroum et al.
investigational drug is a potent CYP3A4 inhibitor, not all substrates of this

enzymes need to be tested to warn against an interaction with this drug.
The following are examples of appropriate labeling language
recommended by the FDA guidance:
Drug-Drug Interactions, Clinical Pharmacology
X In vivo metabolic drug-drug interaction studies indicate little or no
pharmacokinetic effect:
Data from a drug-drug interaction study involving (drug) and (probe
drug) in____ patients/healthy individuals indicate that the PK disposition of
(probe drug) is not altered when the drugs are co-administered. This
indicates that (drug) does not inhibit CYP3A4 and will not alter the
metabolism of drugs metabolized by this enzyme.
X In vivo metabolic drug-drug interaction studies indicate a clinically
significant pharmacokinetic interaction:
The effect of (drug) on the pharmacokinetics of (probe drug) has been
studied in____ patients/healthy subjects. The Cmax, AUC, half-life, and
clearances of (probe drug) increased/decreased by ____% (90% Confidence
Interval: ____ to ____ %) in the presence of (drug). This indicates that
(drug) can inhibit the metabolism of drugs metabolized by CYP3A4 and can
increase blood concentrations of such drugs. (See Precautions, Warnings,
Dosage and Administration, or Contraindications sections.)
Precautions and/or Warnings
X An interacting drug causes increased concentrations of the substrate
but the administration of both drugs may continue with appropriate
dosage adjustment. Results of the studies are described in Clinical
Pharmacology, Drug-Drug Interactions, Precautions and/or Warnings and
may state:
Drug____/class of drug causes significant increases in concentrations of
____ when co-administered, so that dose of ____ must be adjusted (see
Dosage and Administration). If there is an important interaction,
information for patients should point this out also.
X An interacting drug causes increased risk because of increased
concentrations of the substrate and the interacting drug should not be used
with the substrate. After describing the interaction in the Clinical
Pharmacology section, there should be a Contraindications section and
possibly a boxed warning if the risk is serious.
Drug____/class of drug can cause significant increases in concentrations
of drug____ when co-administered. The two drugs should not be used
together.
Dosage and Administration
concentrations of the substrate, but the administration for both drugs may
continue with suitable monitoring: Drug____/class of drug leads to

significant increases in blood concentrations of ____ by____%. The dose of

____ should be decreased by ____% when the patient is also taking ____.
Patients should be closely monitored when taking both drugs.
Contraindications
concentrations of the substrate and should not be co-administered:
Drug____/class of drug leads to significant increases in blood concentrations
of ____, with potentially serious adverse events. Administration of ____ to
patients on drug____/class of drug is contraindicated.
European Guidance on the Investigation of Drug Interactions

The European Agency for the Evaluation of Medicinal Products issued in
December 1997 a note for guidance on the investigation of drug interactions
[98]. This guidance took effect in June 1998. Unlike the FDA guidance
which only dealt with the in vivo metabolic aspects of drug interactions, the
European guidance covered both pharmacodynamic and pharmacokinetic
drug interactions (absorption, distribution, and elimination both at the
renal excretion and hepatic/biliary levels as well as changes in blood flow).
The European guidance makes certain recommendations that are either
not covered or sligthly differ from the FDA recommendations. These
recommendations are as follows:
A. The need for a pharmacodynamic interaction study should be
determined on a case by case basis taking into account the following points:
1. When the drugs likely to be co-administered have similar

mechanisms of action or potentially similar interaction
mechanisms.
2. When drugs likely to be co-administered have similar or opposing
pharmacodynamic effects.
B. In vitro studies may be helpful in investigating the transport mechanism

and whether a drug is a substrate or an inhibitor of P-glycoprotein.
However, the guidance recommends that potential interactions at this level
be confirmed by well-designed in vivo studies since current in vitro studies
have shown to be of limited value in predicting the magnitude of the
interaction.
C. Displacement interaction studies should be performed when the
investigated drug:
- Has nonlinear protein binding.

- The volume of distribution is small.
- Has a narrow therapeutic index.

334 Marroum et al.
- Is highly bound (>95%) to plasma proteins at therapeutic

concentrations.
- Occupies most of the binding sites (such as when the plasma
therapeutic concentrations at the highest recommended dose
exceed the plasma binding capacity).
- When the investigated drug is administered intravenously and
possesses a high metabolic extraction ratio.
- Displacement studies should probably be done in vivo, since the
metabolites may also be involved in the interaction. If the studies
are performed in vitro, then the possible contribution of the
metabolites should also be considered.
D. In general, the guidance recommends conducting an in vitro or in vivo

metabolic interaction studies for metabolic pathways responsible for 30%
or more of the total clearance. However, if toxic/active metabolites are
formed by minor metabolic pathways, the effect of co-administered
inhibitors or inducers of these pathways should also be investigated.
E. Subjects participating in metabolic in vivo interaction studies should
be appropriately genotyped and/or phenotyped if any of the active enzymes
mediating the metabolism are polymorphically distributed in the
population.
F. For inducers or inhibitors, steady-state conditions should be achieved
whenever possible. Approved therapeutic dose regimens should be used in
these studies.
Canadian Guidance on Drug-Drug Interaction Studies

The Therapeutic Products Program of the Canadian Health Agency issued a
guidance document in May of 2001 entitled “Drug-Drug/interactions:
Studies In Vitro and In Vivo” [99]. This guidance as the title indicates covers
both in vitro and in vivo studies. Since the recommendations that are given
in this document do not differ from the recommendations of the U.S. FDA
guidances on this topic, this guidance will not be discussed in detail in this
chapter.
However of interest is this guidance recommendation on how to report
the findings of these studies in the product monograph.
According to this guidance all documented and anticipated drug
interactions should be included in the “Drug Interactions” subsection of the
“Precautions” section with appropriate cross references to other sections of
the label. Drug interactions should be presented as contraindications if they
have the capacity to be life-threatening, cause permanent damage, or elicit
other reactions that would prohibit concomitant administration. Interac-
tions having the potential to cause serious or severe consequences that are

reversible or not life-threatening should normally be included in the

“Warning” section together with recommendations for appropriate risk
management measures. Drug interactions of unknown clinical significance
or resulting in adverse effects that are merely bothersome can generally be
adequately dealt with in the “Drug Interactions” subsection of the
“Precautions” section.
In addition when describing the results of in vivo clinical drug interaction
studies, the monograph should indicate the number of subjects studied, and
whether they were healthy volunteers or patients. The dose and duration of
treatment should also be described.
Drug interactions identified through population pharmacokinetic
approaches, clinical trial case reports, or spontaneous postmarketing
adverse event reporting should be identified as such. The guidance
recommends that in cases where sufficient information is available
comments on the mechanism of the interaction, the clinical manifestations,
as well as actions to prevent or respond to an interaction should be
provided.
As for class labeling, the guidance recommends that manufacturers not
wanting a class labeling with regard to drug interactions should submit data
showing that the possibility of such interactions with their products has
been adequately investigated and dismissed.
ROLE OF POPULATION PHARMACOKINETICS IN THE

STUDY OF DRUG INTERACTIONS
Collecting sparse sampling during the larger phase III clinical trials can
help identify both the intrinsic and extrinsic factors that might affect
exposure to a drug. Thus using such a screening approach might be
valuable in detecting unsuspected drug-drug interactions especially in
patients exhibiting a higher incidence of side effects. Both the U.S. FDA
guidance and the Canadian guidance state that a well-executed
population analysis can provide further evidence of the absence of a drug
interaction when in vitro data suggest the lack of one. However, on the
other hand both guidances agree that the sparse sampling approach to
detect a drug interaction is not yet well established and that it is unlikely
that one will be able to rule out an interaction that is strongly suggested
by information that is obtained from in vitro or in vivo studies
specifically designed to detect an interaction. This is due to the presence
of confounding variables that are not controlled in the study that reduce
the power to detect an interaction. The major advantage of such an
approach is that the study is conducted in the target patient population
and thus clinical inferences on the magnitude of the interaction as well as

336 Marroum et al.
dosing recommendations are easier made from the results obtained.

Another advantage of such an approach is that it does not expose
healthy volunteers to unnecessary side effects of the drug. However,
these studies are considered to be much more difficult to perform and
believed by some to be more costly [100, 101].
CONCLUSION
There is an increased awareness both by the regulatory authorities and by

drug sponsors on the importance of the elucidation of the potential for drug
interactions of a new molecular entity. Establishing the drug interaction
profiles of a new drug and providing proper information on dosing
recommendations when certain drugs are given together is an important risk
management tool and will go a long way in avoiding unwanted adverse
events.
A well-designed program that takes into account the available in vitro
technologies, the right in vivo studies, the appropriate model compounds
and a population screen during the phase III trials will not only provide the
necessary information that is required by regulatory agencies but will also
provide guidance to the prescriber and patient on the appropriate dosing
recommendations when multiple drugs are co-administered [102].
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102. Huang, S.M.; Honig, P.; Lesko, L.; Temple, R.; Williams, R. An Integrated
Approach to Assessing Drug-Drug Interactions: A Regulatory Perspective.
Drug-Drug Interactions, Rodrigues, A.D., Ed.; Marcel Decker: New York,
2001, 605–632.

15
Assessing the Effect of Disease State
on the Pharmacokinetics of the Drug
Marie Gårdmark, Monica Edholm,

Eva Gil Berglund, Carin Bergquist,
and Tomas Salmonson
Medical Products Agency
Uppsala, Sweden
INTRODUCTION
Efficacy and safety of a new medicinal product are established in phase III
trials conducted in a selected group of patients. In fact, with the aim to
reduce the variability, there have been an increasing number of inclusion
and exclusion criteria imposed in the phase III studies submitted to the
Medical Products Agency in Sweden over the last 10 years. However, when
approved, the product is often used in a wider group of patients. To
compensate for this discrepancy the pharmaceutical industry and regulators
use pharmacokinetic data, together with studies in animals, to identify
subgroups of patients where the exposure is changed to an extent that they
should not be treated with the medicinal product, or the dose needs to be
adjusted. The aim of this chapter is to discuss disease states that may
influence the pharmacokinetics of a medicinal product. References are made
345
346 Gårdmark et al.
to a number of regulatory guidelines. It is, however, important that these are

considered to be guidelines and nothing more than guidelines. Each new
drug has its own characteristics and should be developed according to
current scientific standards.
METHODOLOGICAL ASPECTS
The impact of disease on the pharmacokinetics can be evaluated either in

specific studies or by population pharmacokinetic analysis of data from phase
II–III studies. However, the many inclusion and exclusion criteria in today’s
phase III studies may limit the possibility to use a population approach. Such
an approach requires that a sufficiently large number of patients with
different degrees of dysfunction are included in the study, otherwise the results
are of limited value. When sufficient data are available, results from
population analysis alone are fully sufficient for labeling purposes.
When designing or assessing a study in a specific patient population,
there are often a number of pharmacokinetic issues that need to be
considered, including:
• Relationship between concentration and response (both
desirable and undesirable effects) i.e., how much can the
concentration change without influencing the efficacy or safety
of the drug.
• Given the intended therapeutic use of the drug, what is the major
concern: concentration-dependent side effects or lack of
efficacy?
• Variability in the population (are outliers cause for concern?)
• Is it reasonable to assume that the pharmacodynamics is the
same in different subpopulations?
Obviously, the answers to the questions above and the selected study design
should be based on the pharmacokinetic/pharmacodynamic characteristics
of the drug. The additional issues that need to be considered include:
• Are there any nonlinear properties that would justify steady-

state studies? A multiple-dose study is desirable when the drug or
an active/toxic metabolite is known to exhibit nonlinear or time-
dependent pharmacokinetics. Otherwise a single-dose study is
sufficient.
• Dose selection. In single-dose studies, a dose within the
therapeutic dosage range should be used. For multiple-dose
studies, lower or less frequent dosing may be needed to avoid
unsafe accumulation of drug and/or metabolites.

Assessing the Effect of Disease State 347
• Which pharmacokinetic parameters are of greatest concern?

Extent of bioavailability (F) and clearance (CL) are often most
important, usually measured as AUG. When appropriate,
emphasis should also be given to rate of absorption or other
“secondary” parameters such as Cmax.
• Should only the parent compound be measured or should also
the active/toxic metabolites be determined? If the metabolites are
active or toxic, the impact of disease states on these metabolites
should be evaluated. Evaluation of inactive metabolites should
be considered when appropriate.
• Should the pharmacokinetics be based on total or unbound drug?
For example, when plasma protein binding may be altered, the
pharmacokinetics should be described and analyzed with respect
to the unbound concentrations of the drug and active metabolites
in addition to total concentration, unless the drug or metabolites
exhibit relatively low extent of plasma protein binding.
In addition to selecting which trials should be conducted, the sponsor must also
decide when to perform the studies. If available, information on influence of
disease on the pharmacokinetics of the drug could be of value when designing
the phase III programme. On the other hand, there may be financial as well as
ethical reasons to perform these studies late in phase III or even after a regulatory
approval of the medicinal product. In the latter situation, a specific subgroup
may be contraindicated pending availability of this information.
TARGET POPULATION
Introduction
Several factors may induce a difference in pharmacokinetic parameters
between volunteers and target population, such as disease-related factors
and demographic factors (e.g., age, gender, and weight). The rate and extent
of absorption, the extent of distribution and/or the elimination rate could be
altered as a consequence of a disease. The disease is a large source of
variability in drug response between patients and the variability can, at least
in part, be attributed to the pharmacokinetics.
Disease-related pharmacokinetic differences between target patients and
volunteers can largely be explained by functional disturbances of the eliminating
organs, liver and kidney separately discussed later in this chapter. But, even
when renal and hepatic elimination has been accounted for, pharmacokinetic
differences between populations may persist. For a number of disease states,
an effect on the pharmacokinetics is not expected. Examples of such conditions
are pain (at least mild to moderate), mild infections, skin disorders, psychiatric

diseases. Others are more likely to induce a pharmacokinetic change. These

include cardiovascular disorders with effects on perfusion rate, endocrine
dysfunction as diabetes causing reduced renal function and altered protein
binding and severe respiratory disorders that may give hypoxaemia and
disturbance in the acid-base balance. Ultimately, whether or not a significant
disease-related change will appear depends on the pharmacokinetic
characteristics of the drug, e.g., elimination pathways, high- or low-hepatic
extraction, degree of protein binding. So far, there is no specific guideline
addressing pharmacokinetic differences due to disease factors.
Studies in Healthy Volunteers and Patients

The pharmacokinetic characteristics of a drug are usually evaluated in early
studies conducted in, if ethical, healthy volunteers (HV) under well-defined
and controlled conditions. Multiple-dose studies are conducted either in a
selected patient population suffering from the disease for which the drug is
considered to be indicated and/or in HV. In later studies, the
pharmacokinetics in the target population is evaluated using various
approaches, such as gathering full pharmacokinetic profiles in limited
numbers of patients or obtaining few steady-state concentrations
measurements e.g., sparse sampling [2]. From these results, a relation
between the grade of illness and the impact on pharmacokinetic parameters
could be established. However, comparisons between volunteers and
patients are usually confounded by demographic variables, for instance age
or weight, which also have a potential to affect the pharmacokinetics, and
hence such divergence has to be recognized and assessed.
The European guideline, Pharmacokinetic Studies in Man [1], states that
“Studies should be conducted in patients suffering from the disease for
which the drug is claimed to be indicated. If feasible, the relation between
dose, plasma concentration, and effect should be studied. Particularly, it
should be established that the pharmacokinetic behaviour of the drug in
patients corresponds to that in healthy volunteers. The full range of
pharmacokinetic studies needs only be repeated in patients if studies
indicate that the pharmacokinetics in this group differ from those in healthy
volunteers.” The last sentence leaves the subject open for interpretations,
since the word “differ” has not and cannot be defined quantitatively. If an
important difference is detected it is still questionable whether all studies
have to be repeated. Instead, the number of studies necessary should be
judged on a case-by-case basis depending on the degree and type of
difference and also the general characteristics of the drug. If there is reason
to believe that certain physiological or pathological factors, such as certain
functional or anatomical disorders of the gastrointestinal tract, might
substantially alter absorption, separate pharmacokinetic studies in suitable

volunteers or patients could be performed. Information about

pharmacokinetic differences between healthy and patient populations
should be included in the labeling of the drug.
For drugs displaying marked pharmacokinetic divergence between
populations, predictions based on HV data might not adequately enough
characterize the target patients. The following issues need to be considered.
• Further studies to evaluate the pharmacokinetics in special

populations, e.g., renal and hepatic impairment, are conducted
in individuals not necessarily suffering from the target disease,
and hence reducing the predictability. Moreover, healthy
volunteers are often chosen as the control group, whereas the
target population would be a more appropriate control group,
given a difference in pharmacokinetics.
• Conventional interaction studies are often conducted in healthy
volunteers and the results cannot always be extrapolated to the
target population. For instance, there might be disease or
demographic-related factors affecting the drug absorption
differently in the target population, increasing or decreasing an
interaction on bioavailability. Furthermore, the patient might
use concomitant therapy that is not taken into account in the
volunteer study.
• Usually, the interindividual variability in pharmacokinetic
parameters is lower in healthy volunteers compared with the
more heterogeneous target population. Thus, mean parameter
estimates could be comparable, but there could still be
unexpectedly high incidences of adverse events or therapeutic
failure in some patients due to too high or low drug levels,
respectively. In addition, the variability in a parameter between
occasions might be higher in patients because of disease
progression factors.
• Healthy volunteers or selected patients are included in early
clinical studies, in which the first pharmacokinetic data (and
sometimes PK/PD) in man is evaluated. These results are then used
as support for dose selection in later phases, which might result in
less suitable dosing regimens, given that there is considerable
pharmacokinetic difference between volunteers and patients.
To assess the influence of a disease, mean parameter estimates or

concentrations/exposure and their corresponding variability in volunteers
should be compared with estimates from the patient population. However,
when comparing results from separate studies (phase I vs. phase III) there
might be confounding factors such as demographic dissimilarities, that

should be taken into account, e.g., age or gender differences. Phase II trials
often include highly selected patients, which might not reflect the proper
target population. One problem arises when the phase III trial in the target
population has not been designed to estimate pharmacokinetic parameters,
but supply, e.g., single trough concentrations, along with mixed inter- and
intra-individual variability estimates. In these cases, comparisons are less
reliable since the trough levels could be influenced by different dosing
regimen, sampling, and assay error and may not represent the “true”
concentrations [2]. If data from volunteers and patients are pooled,
important patho-physiological factors can be included as covariates.
Subgroups suffering from additional diseases (e.g., obesity) could be
separately analyzed and compared with the total population, but the
number of subgroup patients needs to be sufficiently large.
Impact of Comorbidity
The pharmacokinetics of the drug is evaluated in the target population
fulfilling the criteria for which the indication is sought. The target
population may be very wide and include subpopulations suffering from
additional diseases affecting the pharmacokinetics of the drugs. These
patients might not at all be represented in the trials or in too low numbers,
not allowing their altered pharmacokinetic characteristics to be detected.
It would be useful to know the kinetics of drugs in a very large number of
patho-physiological situations; however, it is clear that this knowledge
requires multiple, long, and expensive studies, which cannot all be
performed. Examples of therapeutic areas for which the intended
population is wide and difficult to fully incorporate in the usual clinical
trials are pain medications, antihypertensive drugs, and antibiotics. If
important disease-related effects on the pharmacokinetics are detected for
a certain patient population, the information should be included in the
labeling and, if necessary, appropriate restrictions such as
contraindication, warnings, or dose adjustment should be included in the
labeling.
Examples
Altered pharmacokinetic characteristics have been reported in the literature
for various diseases or conditions, some of which are briefly summarized
below.
Circulatory Disorders. This term includes, for example, congestive
heart failure and malignant hypertension, generally characterized by
diminished organ perfusion. Acute cardiovascular failure reduces the
perfusion of liver and kidney and hence CL of highly extracted drugs

might be affected. The enteral absorption may be reduced due to

diminished perfusion and occasionally increased back pressure on the gut.
The volume of distribution might be increased. The kinetics of distribution
is affected, with diminished perfusion rates to certain organs. Reduced
perfusion may cause metabolic acidosis that can alter the distribution of
ionized drugs [3, 4].
Obesity. Obese individuals are subjected to different drug treatments
for which the dosage recommendations have not been specifically
evaluated with respect to obesity. Obesity is likely to affect drug
distribution and elimination, whereas absorption is less likely to be
modified. Alterations that may occur in obesity are increased
distribution volume due to drug tissue distribution, alteration of the
drug metabolic activity and cardiac performance. It has turned out to be
rather difficult to predict the impact of obesity based on its lipophilic
characteristics when it comes to markedly lipophilic drugs, whereas
more hydrophilic drugs are more predictable, possibly due to their
distribution mainly to lean tissues. For a more lipophilic drug, changes in
distribution volume might appear and then adjusting the loading dose to
bodyweight should be considered [5].
GI-Disorders. Diseases in the GI-tract may affect different factors
important for drug absorption, and the effect on the overall
pharmacokinetics is not always predictable. Inflammatory bowel diseases,
such as Crohn’s or ulcerative colitis, affect the absorption surface area and
there are several reports on altered absorption in patients suffering from
these conditions [6]. In celiac disease, associated with stunted small
intestinal villi and alteration of gastric emptying and pH, the intestinal
CYP3A4 content was decreased [7]. Changes in pH (e.g., achlorhydria or
AIDS gastropathy) might delay and reduce the absorption of pH-dependent
drugs such as ketoconazole [6]. Changes in GI-motility, by e.g., irritable
bowel syndrome (small intestine), diabetes mellitus and nonulcer dyspepsia
(stomach), and idiopathic constipation (colon), may affect the absorption of
orally administered drugs by changing the rate of delivery, bioavailability, or
mucosal absorption. For poorly absorbed drugs both the rate and extent of
absorption are likely to be altered, whereas for well-absorbed drugs an
effect is mainly seen on the rate of absorption. Predictions are, however,
complicated by factors such as drug-related properties, the formulation, and
food effects [8].
Surgery. Some drugs are intended for postoperative treatment and hence
the dosing recommendations are evaluated in the same population.
However, also drugs unrelated to the surgery are used postoperatively, such
as cardiovascular drugs. Absorption, distribution, and elimination of drug
might be altered due to diminished gastric emptying, altered protein binding
and renal impairment [9].

Cystic Fibrosis. In patients with CF the absorption rate varies but the
extent of absorption is generally not altered. There is a difference in
distribution volume due to reduced lean body mass. Patients with CF
have been associated with increased metabolic CL of many drugs.
Increased activity of both phase I and II reactions have been
demonstrated, although not all CYP-isoforms were affected. The renal
CL of many drugs is enhanced, although no mechanistic explanation has
been found [10].
Organ Transplantation. Following transplantation, patients undergo
marked changes in the physiological functions associated with the
transplanted organ. Drug absorption, distribution, and elimination may
undergo time-dependent transition from that associated with organ failure
to that of the normal state. A thorough understanding of how the
pharmacokinetics is influenced is essential for optimal drug therapy and for
improvement of long-term survival [11]. For sirolimus, indicated for
prophylaxis of organ rejection in patients receiving a renal transplant, oral
clearance was reduced and half-life prolonged in the patient population.
The distribution volume was lower in patients as was also the blood to
plasma partition ratio (data on file).
Conclusion
Disease-related differences in pharmacokinetics may give rise to
exposure differences between volunteers and patients and may be
responsible for part of the inter- and intra-individual variability within
the target population. The importance of any pharmacokinetic changes
is related to the therapeutic index of the drug and thus the therapeutic
consequences of altered pharmacokinetics should be considered. The
probability of a change in any pharmacokinetic parameter might be
considered, e.g., the bioavailability may or may not be sensitive to a
difference in absorption characteristics. If relevant changes are found
and deemed as therapeutically important, these should be considered
when designing and evaluating studies from which pharmacokinetics in
HVs are extrapolated to patients, e.g., renal- and hepatic-impairment
and interaction studies.
It is not possible to cover all patients in the clinical trials that in the future
possibly will use the drug. Therefore, the only studies that should be
submitted before marketing are those that seem necessary with regard to
properties, indications, contraindications, routes of elimination, scheme of
administration of the drug, and those required to define the necessary dose
changes that cannot be calculated from the pharmacokinetic parameters
available from HV and in patients without functional disturbance of
absorption, distribution, and elimination systems [1].

RENAL INSUFFICIENCY
Introduction
Renal excretion of drugs involves filtration, secretion, and reabsorption.
The unbound fraction of a drug is filtered in the glomerulus. Also small
proteins are freely filtered, but when the molecular weight of the protein
exceeds 20,000 g/mole filtration falls sharply and filtration of albumin
(molecular weight 69,000 g/mole) is very limited [3]. The filtration can be
calculated by fu*GFR, where fu is the fraction unbound in plasma and
GFR is the glomerular filtration rate, which in a 70 kg, 20-year-old man is
about 120 ml/min. Drugs may also be secreted by active transport systems.
These are predominantly located in the proximal tubule. If renal clearance,
CLR, exceeds the filtration (CLR>fu·GFR), both secretion and reabsorption
may be involved, but secretion is more pronounced. Reabsorption is
higher than secretion if renal clearance is less than the filtration (CLR<
fu·GFR). For the majority of exogenous compounds, reabsorption occurs
by a passive process. Reabsorption occurs all along the nephron, although
the majority is reabsorbed in the proximal tubule. Many proteins,
especially low molecular weight proteins, are substantially filtered in the
glomerulus, but not excreted in urine. These are metabolized by enzymes
located in the brush border of the proximal tubule lumen. Catabolism of
proteins continues until constituent amino acids are formed.
As described above, renal function consists of several mechanisms.
These may be differently affected by factors that influence renal function,
e.g., age and renal disease. In adults, renal function steadily decreases with
age, starting by the fourth decade [12]. Both glomerular number and size
decrease with increased age [13]. Glomerular filtration rate and tubular
function are generally considered to decrease at a parallel rate with age
[12, 14].
Effects of Reduced Renal Function on Pharmacokinetic

Parameters
The excretion of many drugs can be affected by the presence of renal
disease, and for drugs principally eliminated via the renal route, drug
excretion is diminished in patients with reduced renal function. Reduced
renal excretion is not the only change in drug disposition in patients with
renal insufficiency. There may be changes in absorption, protein- and tissue
binding, and distribution and hepatic metabolism [15]. In addition,
pharmacodynamics may also be altered in renal impairment [16].

Absorption and Bioavailability

Altered drug absorption may be a result of prolonged gastric emptying time
and increased gastric pH [15]. Increased bioavailability has been reported in
patients with renal insufficiency secondary to decreased first-pass
metabolism.
Distribution
Changes in drug distribution may arise from either fluid retention or
changes in the extent of protein binding in tissue and plasma [15]. The
plasma protein binding of most acidic drugs is decreased in uraemic patients
[17]. These drugs are often highly bound to albumin and any modifications
in the binding may have large effects on the fraction unbound. The
decreased protein binding may be caused by hypoalbuminaemia,
accumulation of endogenous competitive displacing substances or
decreased affinity of human serum albumin caused by alteration in the
conformation or structural arrangement of albumin-binding sites [17].
Conversely, the protein binding of basic drugs may be differently affected in
renal failure (increased, decreased, or unchanged binding) [15, 18].
Metabolism
The results of studies on the effect of renal impairment on hepatic drug
metabolism are conflicting. Metabolism has been shown to be increased,
decreased or be unaffected by renal failure [18, 19]. Different drugs
metabolized by the same cytochrome P450 isoenzyme have been reported to
be differently affected by renal impairment. For different beta-blockers
metabolized by CYP2D6, metabolism has either been reported to be
decreased or unchanged, and for different calcium channel-blockers
metabolized by CYP3A4, metabolism has been reported to be increased,
decreased, or unchanged [18]. Sulphatation and glucuronidation are
generally normal, whereas N-acetylation of isoniazid has been reported to
be reduced in chronic renal failure [15, 20]. Metabolic ratios of metabolite
and drug excreted in urine are often used for phenotyping of polymorphic
drug metabolizing enzymes as well as for estimations of enzyme activity. As
renal clearance of the drug or metabolites may affect such ratios, the ratios
may be different in patients with renal impairment than in the overall
population.
The pharmacokinetics of drugs metabolized or catabolized in the
kidneys, but not excreted in urine, such as peptides and small proteins, is
affected by renal impairment. The elimination of these will be decreased,
resulting in accumulation in renal impairment.

Accumulation of Metabolites
Metabolites that are renally excreted will accumulate in renal impairment.
This could lead to increased efficacy or toxicity for pharmacologically
active or toxic metabolites. Also metabolites that are considered relatively
inactive in patients with normal renal function may reach active/toxic
levels if the accumulation of the metabolites is extensive in renal
impairment.
Estimation of Renal Function

Renal function is usually assessed through calculation of glomerular
filtration rate (GFR). The reference method for estimating GFR is inulin
clearance. Inulin is an inert polysaccharide cleared exclusively by glomerular
filtration. The method includes constant intravenous infusion of inulin and
timed collection of urine and is not practical for routine clinical purposes. A
number of alternative methods have been developed for estimation of GFR.
Many involve collection of urine and may give inaccurate results unless
collection of urine is complete, including complete emptying of the bladder.
Several methods to determine the plasma clearance of a suitable exogenous
marker have been developed. These include radionuclides such as 51Cr-
EDTA and 99mTc-DTPA (diethylenetriaminepentaacetic acid) [21]. Although
these methods are accurate, the requirement of radiolabeled tracers
complicates the procedure (complicated handling, storage, and disposal of
waste) and excludes certain patients, such as pregnant women. Alternative
nonlabel filtration markers include the exogenous markers iothalamate and
iohexol [22, 23] or endogenous markers such as Cystacin C [22] and, most
importantly, creatinine [24].
GFR can be estimated by calculating creatinine clearance (CLcr)
utilizing the serum creatinine concentration (Scr) and other patient
characteristics such as bodyweight, age, gender, and height. All methods
for estimating CLcr from Scr are simple, but are limited. Prediction of
CLcr will not be accurate unless renal function and serum creatinine are at
steady state and is not accurate in patients with unusually low or high
muscle mass, in patients with marked obesity or ascites [24], or in patients
with liver disease [25]. Moreover, creatinine is not exclusively filtered, but
also subject to tubular secretion. Thus, GFR is overestimated by CLcr.
This is especially evident in severe renal impairment. Creatinine clearance
can also be determined from serum creatinine concentration and urinary
excretion of creatinine. With this method, some of the drawbacks of using
Scr can be avoided. The results are more accurate than estimation from Scr
alone if complete collection of urine, including complete emptying of the
bladder, can be obtained.

Given the limitations of using CLcr as a measure of renal function, more

accurate methods for measuring renal function, such as 51Cr-EDTA,
iothalamate or iohexol, should be considered in clinical studies evaluating
the influence of renal function on the pharmacokinetics of new drugs.
Classification of Renal Impairment
Renal function is usually classified as normal renal function (CLcrⱖ80 mL/

min), mild renal impairment (ⱖ50-<80 mL/min), moderate renal
impairment (ⱖ30 mL/min-<50 mL/min), severe renal impairment (<30 mL/
min), and end-stage renal disease (patients requiring dialysis) [26].
However, dose adjustments should be based on the actual results and do not
need to follow the classification.
Evaluation of Pharmacokinetics in Renal Impairment

When and How to Perform Pharmacokinetic Studies in Patients with
Renal Impairment
The FDA guidance for industry “Pharmacokinetics in patients with
impaired renal function—Study design, data analysis, and impact on dosing
and labelling” [26] gives detailed information on the FDA requirements for
when and how pharmacokinetic studies should be performed in patients
with renal impairment. A corresponding European guideline is presently
being written, but has not yet come into operation (2003) [27]. Although
not yet formally specified in an approved guideline, the requirements in
Europe for pharmacokinetic characterisation in patients with renal
impairment are essentially similar to those of the FDA.
A pharmacokinetic study in patients with impaired renal function is
recommended when renal impairment is likely to significantly alter the
pharmacokinetics of a drug and/or its active/toxic metabolites, and a dosage
adjustment may be needed for safe and effective use in such patients.
As described above, severe renal impairment has been associated with
changes in absorption, hepatic metabolism, protein binding, and distribution
also for drugs that not are excreted renally. Hence, pharmacokinetic
characterization in patients with severe renal impairment should be
considered also for drugs eliminated mainly by metabolism, in particular
when the drug or its active metabolites exhibit a narrow therapeutic index.
Study Design
The primary goal of a study in patients with impaired renal function is to
determine if the pharmacokinetics is altered to such an extent that the

dosage should be adjusted from that established in the phase III trials, where
efficacy and safety has been shown. Thus, the study should focus on
comparing patients with renal impairment with patients with renal function
that is typical of the clinical trial patient population—not necessarily with
healthy young volunteers.
To ensure adequate representation of patients with various degrees of
renal impairment, approximately equal numbers of patients from each of
the renal impairment groups (normal renal function, mild, moderate, severe
renal impairment, end stage renal disease) should be recruited. The renal
function groups should be comparable with respect to age, gender, and weight
and other factors with significant potential to affect the pharmacokinetics of
the drug (e.g., diet, smoking, alcohol intake, concomitant medications,
ethnicity). The number of patients enrolled should be sufficient to detect
clinically relevant pharmacokinetic differences.
If there is good reason to believe that renal impairment does not affect the
pharmacokinetics to a degree sufficient to warrant dose adjustment, it may
be sufficient to study only patients at the extremes of renal function (i.e.,
patients with normal and severely impaired renal function). If the results
confirm that renal impairment does not relevantly alter the
pharmacokinetics, no further study is warranted. If the results do not
strongly support such a conclusion, the intermediate renal function groups
(mild and moderate renal impairment) should also be studied.
A population pharmacokinetic evaluation of patients participating in
phase II/phase III clinical trials may be used to assess the impact of renal
function on the pharmacokinetics of a drug. In principle, such a population
pharmacokinetic study design and analysis can be an acceptable alternative
to a specific renal impairment study if:
• it includes a sufficient number of patients and a sufficient
representation and range of renal function so that the study
could detect relevant pharmacokinetic differences
• unbound concentrations have been measured, when appropriate
• both parent drug and potentially active/toxic metabolites are
measured, when appropriate.
Patients with severe renal impairment are often excluded or poorly
represented in population pharmacokinetic studies. When that is the case
for a drug likely to be administered to such patients, a separate and
complementary study could be conducted to assess the pharmacokinetics in
patients with severe renal impairment (e.g., a study evaluating the
pharmacokinetics in subjects with severely impaired renal function
compared with subjects with renal function typical for the phase III
population). The data from both sources should be used in the overall
assessment of the effect of renal impairment. Even if the above requirements

regarding unbound concentrations and analysis of metabolites cannot be

fulfilled for samples collected in phase II/III studies and the population
analysis cannot replace a conventional study, population analysis of the
impact of renal function on the pharmacokinetics in the target population is
recommended, as this provides valuable information regarding variability in
the target population. These data could be evaluated in conjunction with the
data from a conventional renal impairment study.
Dialysis
Dialysis may significantly alter the pharmacokinetics of drugs. When a
significant fraction of the drug or active metabolite(s) is removed by dialysis,
a change in the dosing regimen may be required, such as a supplementary
dose following the dialysis procedure. It should be remembered that also
drugs that are not excreted by the renal route to a large extent may be
removed by dialysis.
For drugs that are likely to be administered to end-stage renal disease
(ESRD) patients undergoing dialysis and where the drug or active
metabolites are likely to be extracted during dialysis to such an extent that
supplementary dosing after dialysis may be required, evaluation of the
pharmacokinetics under both dialysis and nondialysis conditions should be
considered in order to determine the contribution of dialysis to the
elimination of the drug and potentially active metabolites.
Presentation and Evaluation of Results
In the presentation of results, a graphical description of the relationship
between individual pharmacokinetic parameters and renal function should
be included. This is important for assessment of the variability in normal
and reduced renal function and facilitates the identification of cut-off for
dose adjustment. The FDA guideline emphasises the construction of
mathematical models to evaluate the relationship between renal function
and pharmacokinetic parameters. Although renal clearance of many drugs
decreases with reduced renal function, the relationship between renal function
and pharmacokinetic parameters is not necessarily linear. Descriptive statistics
of the pharmacokinetic parameters according to renal function (normal, mild,
moderate, and severe renal impairment) can also be presented.
Study results including the model for the relationships between renal
function and relevant pharmacokinetic parameters should be used to
construct specific dosing recommendations. Typically, the dose is
adjusted to produce a comparable range of unbound plasma
concentrations of drug or active metabolites in both normal patients and
patients with impaired renal function. As discussed above, it is important
to consider the variability in pharmacokinetics in renal impairment, the
therapeutic index and consequences of reduced and increased exposure,

respectively, in this assessment. A recently proposed approach is to

estimate appropriate cutoffs and doses, given the information on
pharmacokinetic parameters and distribution of renal function in the
population [28]. Regardless of whether specific dose reductions are
recommended or not, simulations of the steadystate exposure and
predicted variability at the proposed dose(s) is a valuable tool for
confirming the suitability of the chosen posology.
Current Experience and Ways Forward

Generally, renal impairment studies are fairly well performed, but there is
room for improvement. Deficiencies still seen in these studies include poorly
presented results and dose adjustments based on mean data without taking
variability into account. There is room for improvement in these areas. Also,
the use of unbound exposure, when applicable, and population
pharmacokinetics is likely to increase. In the future, increased evaluation is
expected of the influence of renal impairment on the pharmacokinetics of
renally excreted metabolites, both active and inactive, and hopefully more
studies will be performed evaluating the effect of renal impairment on active
transporter systems.
FDA has published a survey of renal impairment studies performed
during 1996 and 1997 [29]. The survey indicated that in the past, no
consistent pharmacokinetic property drove the decision to conduct renal
impairment studies, there was no consistency in study design, number of
groups of patients with reduced renal function, and the number of patients/
group. In most protocols 24 h CLcr was used to assess renal function, in
75% of the studies the doses used were in the therapeutic range, a point
estimate with ANOVA was generally used to analyze data and there was no
consistent method for presenting data from renal impairment studies in the
product labeling. In part, based on this survey FDA developed the guidance
for studies of pharmacokinetics in renal impairment to promote
welldesigned studies with adequate presentation of results resulting in
consistency in product labeling.
LIVER DISEASE
Introduction
The pharmacokinetics of drugs may be altered in liver disease. This
primarily applies to drugs that are eliminated by the liver to a substantial
extent although drugs that are eliminated by other organs may be affected
through effects secondary to the hepatic impairment. Possible causes of the
changed pharmacokinetics are several, including reduced enzyme activity,

altered hepatic blood-flow, shunting of blood past the liver, decreased

protein binding and secondary renal impairment. To what extent the drug is
affected by the hepatic impairment is dependent on the pharmacokinetic
properties.
Liver disease is a heterogeneous group of diseases with different
morphological changes and symptoms. Presently there is no optimal marker
for assessing hepatic function. Therefore it is difficult to predict the
pharmacokinetics of a certain drug in individual patients as well as making
extrapolations to nonstudied types of liver diseases. Recently the U.S. FDA
has issued a guidance for pharmacokinetics in patients with impaired
hepatic function [30] and an EU guideline is under preparation [31].
Liver Diseases—A Variety of Conditions

There are numerous reasons for impairment of the hepatic function. In the
western world, chronic alcohol abuse is one of the main causes of liver
disease and can cause steatosis, alcohol hepatitis, and cirrhosis.
Steatosis is a condition caused by disturbances in the lipid metabolism
and produce an accumulation of triglycerides within the hepatocytes. The
condition may appear quickly and is reversible if the cause of the
accumulation is removed. The condition is mainly caused by alcohol but
may also be caused by malnutrition, hepatotoxic substances, diabetes, and
obesity.
Hepatitis is mainly caused by viruses, hepatotoxic substances, and
autoimmune diseases. The condition is characterized by cell necrosis and
inflammation in the liver. All forms give the same alterations of the liver,
including simultaneous necrosis and degeneration of hepatocytes,
infiltration of mononuclear cells, degeneration of Kupffer cells, and varying
degree of cholestasis.
Cirrhosis is not a disease in itself but a stage in the course of
inflammatory liver diseases. Cirrhosis can be caused by liver damage
resulting from alcoholism, hepatitis B and hepatitis C, drugs, metabolic
disorders, prolonged cholestasis, etc. Cirrhosis is characterized by increased
presence of fibrous tissue, destruction of the lobular architecture and
sinusoidal network, and nodular degeneration. The hepatic synthesis of
proteins such as albumin, prothrombin, and enzymes is decreased. Cirrhosis
often gives rise to portal hypertension. In portal hypertension, the blood
flow coming from the intestine through the liver via vena porta is reduced
while the arterial blood flow is increased relative to the portal flow. Many
cirrhotic patients have portacaval shunts, where a substantial fraction of the
portal blood bypasses parenchymal tissue in the liver or enters directly into
the superior vena cava via esophageal varices. A characteristic late sign of
liver disease is ascites, an accumulation of extracellular fluids in the lower

abdominal area. Ascites is believed to be caused by a combination of portal

hypertension and decreased colloid osmotic pressure in the blood. The
degree of portal hypertension, shunting, ascites, and residual metabolic
capacity varies between cirrhotic patients.
Usually pharmacokinetic studies are performed in cirrhotic patients. As
there is no optimal marker(s) for assessing hepatic function, extrapolation
from study results to individual cirrhotic patients as well as to patients not
having cirrhosis is problematic.
Estimates of Liver Function

Child-Turcotte classification is an empirical but commonly accepted way to
estimate the grade of cirrhosis even though it is not known to what extent it
may be used to estimate hepatic function. In 1973, Pugh used Child’s
classification system but added the prothrombin time when he wanted to
classify patients in a study with regard to the risk related to surgery of the
oesophagus [32]. Since then, the degree of liver dysfunction is determined
mainly by ranking the patient according to the Child-Pugh classification.
Using this classification, the patients are grouped into mild, moderate, or
severe impairment based on both two clinical symptoms of liver disease
(encephalopathy and ascites) and three clinical chemistry parameters (S-
albumin, S-bilirubin, and prothrombin time) (Table 1).
Based on the Child-Pugh scores, the patients are divided into groups
called A, B, C, or “Mild”, “Moderate”, or “Severe” corresponding to 5–6,
7–9, and 10–15 scores, respectively. As a result, patients with a normal
hepatic function are given a total score of five points and would
consequently be classified as having mild liver impairment.
In the majority of pharmacokinetic studies, the Child-Pugh
classification is used to assess the degree of liver function impairment. In
patients evaluated for classification purpose, it is important that impaired
hepatic function and not some other underlying disease is the cause of
alterations in the Child-Pugh components. For example, in patients with
metastatic cancer, hypoalbuminemia, encephalopathy, and ascites may be
related to cancer cachexia or cancer metastatic to the brain or peritoneal
surfaces rather than impaired hepatic function. The Child-Pugh
classification is not an optimal estimate of liver function with respect to
drug elimination capacity and research is presently going on trying to find
alternative markers. Several markers including antipyrine, trimethadione,
caffeine, lidocaine, midazolam, etc., have been tried. Trimethadione, for
example, has been used in the clinic for assessing the function of the liver
before and after liver-transplantation [33]. Such a marker may be a useful
tool for dose-adjustments and could be used alone or in parallel with the
Child-Pugh classification. Until better markers have been found, the

TABLE 1 Ranking of Liver Dysfunction According

to the Child-Pugh Score
Child-Pugh classification system can be used to categorise the degree of

hepatic impairment of patients included in a pharmacokinetic study and
can, together with its individual components, be used when evaluating the
pharmacokinetic results.
To make the dose-adjustments more precise, attempts could be made to
find a clinically available marker that is better correlated with the exposure
(AUC and Cmax) than the Child-Pugh classification. Below is an example
where we tried to correlate the observed exposure not only to Child-Pugh
score but also to the separate clinical chemical parameter included in this
classification system (Figs. 1 and 2). S-albumin was the parameter that was
best correlated with exposure (AUC) of drug. It is, however, recognized that
S-albumin is affected also by other conditions and is not useful as the only
estimate of liver function in patients.
Extrapolations from Cirrhosis to Other Liver Diseases

Although liver disease is a heterogeneous group of diseases, the
pharmacokinetics of a new drug is often limited to studies in cirrhotic
patients. As this is the most common liver disease, this is of benefit for the

FIGURE 1 AUC of an antiinflammatory drug in patients with different degrees of

liver function.
majority of patients. However, extrapolating to patients with other liver

diseases may be difficult. Different kinds of liver diseases may affect the
pharmacokinetics of a drug differently. For example cholestatic and
noncholestatic cirrhosis appear to affect the enzyme expression and/or
availability of specific enzymes in different ways [34, 35]. The amount of
FIGURE 2 Difference in AUC (%) in patients with liver impairment as compared

with matched healthy volunteers vs. S-Albumin.

CYP1A2 appears to be decreased in both hepatocellular and cholestatic

cirrhosis while the levels of CYP3A were only observed to decrease in
patients with hepatocellular cirrhosis. The levels of CYP2E1 were reduced
in patients with cholestatic cirrhosis while the decrease was seen at mRNA-
but not protein-level in livers of patients with hepatocellular cirrhosis [34].
In contrast, markedly (5–10-fold) increases in CYP2E1 levels have been
observed in alcoholics [36, 37]. Due to the discrepancies in effects of the
different diseases, it is important to give information in the labeling
regarding which population has been studied. If new markers of liver
function are found, a safer and more precise extrapolation from cirrhosis to
other diseases could be possible.
Effects of Impaired Liver Function on Pharmacokinetic

Parameters
Presently, hepatic extraction and clearance are usually assumed to proceed
according to the “well-stirred model” [3]. In this model, the liver is assumed
to work as a well-stirred compartment where the drug and enzymes are
evenly distributed. When predicting how the pharmacokinetics is affected
by altered physiological conditions, it should be remembered that a
simplified model of the liver is used.
Altered Hepatic Blood Flow

The hepatic blood-flow may be decreased in cirrhosis but predictions of the
effect on pharmacokinetic parameters are ambiguous. Reduced blood flow
and shunting can both increase the bioavailability of drugs subject to
hepatic first pass metabolism and also reduce the systemic hepatic clearance
of drugs depending on their extraction ratio [3].
Alterations in Enzyme Activity

Drug metabolism catalyzed by cytochrome P450 enzymes is generally decreased
in cirrhosis whereas it may, at present, be less predictable and have been less
investigated in other liver diseases. The reduced metabolism in cirrhosis is
probably due both to reduced viable cell-mass and as well as reduced enzyme
synthesis in the hepatocytes. Decreases have been observed in mRNA-level
and protein-level as well as in enzyme activity [34, 35]. The sensitivity to liver
disease appears to vary between enzymes [38]. Drug metabolism catalyzed by
CYP2C19 appears to be markedly decreased in patients with cirrhosis while
the CYP2D6 activity seems less affected [39]. In general, the UDP-glucuronosyl
transferase enzymes appear less affected than the cytochrome P450 enzymes
although the sensitivity to liver disease differs between isoforms [40, 41]. The

pharmacokinetic consequences of a decrease in enzyme activity depend on the

pharmacokinetic characteristics, e.g., extraction ratio and contribution of liver
metabolism to elimination of the drug [3].
Altered Plasma Protein Binding

Due to a depressed synthesis of albumin in the liver, the protein binding of
drugs may be decreased in cirrhosis. This may have consequences both for
the elimination and the distribution of drugs. The magnitude of the effect on
elimination is again dependent on the pharmacokinetic characteristics of the
drug [3].
Secondary Renal Failure

During the clinical course of cirrhosis, secondary changes in the kidneys
may give rise to renal insufficiency. The renal perfusion can be decreased
and the reabsorption of sodium in the proximal tubule is increased in
decompensated cirrhosis. In addition, in patients with decompensated
cirrhosis, serum creatinine and creatinine clearance estimated from serum
creatinine are not sensitive markers for renal function and often over-
estimate actual GFR [42]. This may be caused by a reduced hepatic
production of creatine, the precursor of creatinine, or a reduced conversion
of creatine to creatinine due to decreased muscle mass [43].
Evaluation of Pharmacokinetics in Hepatic Impairment

The effects of liver disease on the pharmacokinetics of a drug should be
investigated if hepatic metabolism or excretion contributes to a substantial
part of the total elimination and/or if an active metabolite is formed or
eliminated by the liver. In addition, studies may be considered if the drug is
extensively protein-bound or if it has a narrow therapeutic range. The main
objective of a hepatic impairment study is to identify patients at risk and,
when appropriate, to develop dosing recommendations in the patients with
hepatic disease.
The effect of liver disease on the pharmacokinetics of a drug is usually
investigated in cirrhotic volunteers. The diagnosis should, if possible, be
established by biopsies. The group of cirrhotic volunteers should generally
cover the whole range of metabolic impairment. A control group should be
included representing the target population with respect to demographic
factors. The hepatic function groups should be comparable with respect to
age, gender, weight, and other factors with significant potential to affect the
pharmacokinetics. The use of historical controls instead of including
controls with normal liver function is not recommended as, due to

interstudy variability, this may mask a difference in pharmacokinetics of the

drug. The number of volunteers or patients included should be sufficient to
detect clinically relevant pharmacokinetic differences.
Patients classified by the Child-Pugh system as having mild
impairment could have a normal hepatic function and for the majority of
drugs, clinically significant differences are more likely to be observed in
patients with moderate and severe impairment. Thus, a reduced design
including only patients with moderate impairment and controls may be
used to screen for significant effects. If a significant effect is detected in
the moderate group, the pharmacokinetics in patients with mild
impairment needs to be evaluated to propose dose recommendations for
this group.
An alternative way of assessing the effect of liver disease on the
pharmacokinetics of a drug is to use population pharmacokinetic data
obtained in phase II and III studies as has been described for renal
impairment. However, this approach may prove more difficult here due to
e.g., lower prevalence of hepatic impairment in the general population. In
these studies, patients with hepatic impairment should be identified and
classified using the same criteria as has been discussed for the conventional
studies. Population analysis for this purpose should be prespecified.
For prodrugs (i.e., drugs with activity predominantly due to hepatically
generated metabolite), it is possible that the dose may need to be increased,
or the dosing interval shortened, in hepatically impaired patients.
Ways Forward/Room for Improvement

As discussed above, the presently used Child-Pugh classification is not
optimal for assessment of drug-elimination capacity and it would be useful
to find markers better reflecting the different hepatic elimination
mechanisms. Markers like serum albumin, prothrombin time, and bilirubin
may be more related to drug elimination capacity than other components of
the Child-Pugh scale. An ideal marker should be proven relevant and should
preferably not be affected by other conditions. The reason for lack of effect
on the pharmacokinetics may be due to inclusion of subjects in whom,
although classified as having hepatic impairment, the elimination capacity
for the drug is not altered. One way to ensure that the included subjects have
impaired metabolic capacity may be to administer a probe drug (e.g.,
CYP3A4 probe if the compound being investigated is a CYP3A4-substrate)
to confirm that an effect would be detectable in the studied subjects. In the
future, more specific markers may ensure reliable identification of patients
at risk and support proper dosing recommendations to patients with
different degrees of hepatic impairment.

INTERPRETATION OF DATA
Marketing applications for new medicinal products often include studies in

adequate subgroups of patients. The aim is to develop dosing
recommendations that will decrease the overall variability in the population
and ensure that the patient will obtain treatment that is effective and safe.
Factors that should be taken into account are the intended use of the drug,
the pharmacokinetic characteristics of the drug and the PK/PD relationship
regarding efficacy and safety. Based on available information regarding the
latter, target criteria should be specified a priori for what change in the
pharmacokinetics would justify a posology adjustment. The target criteria
should be based on the major concern (side effect or lack of efficacy) for the
specific product. It is not uncommon that not only the mean exposure is
increased in specific subgroups but also the inter-individual variability. In a
group of patients with moderate hepatic impairment, some patients may
show no increase in exposure at all, while others show a significant increase.
Again, what is our main concern—concentration-dependent adverse events
or subtherapeutic level?
When investigating the pharmacokinetics in patients with decreased
organ function the most common approach is to study patients with various
degrees of impairment. To ensure that a sufficient number of patients are
included, patients are often stratified according to predefined criteria into
mild, moderate, and severe impairment. Unfortunately, it is not uncommon
that data are presented as mean values +/- S.D. within these subgroups.
However, when assessing the results from such data, there is no reason to
use these predefined criteria. As has been pointed out above, the entire
information available should be utilized.
In a group of patients with a reduced elimination of the drug compared
with other patients it is often impossible to provide a dosing
recommendation resulting in identical concentration-time profiles.
Regardless of whether the dose is reduced and/or the dosing interval is
increased, one needs to focus on either similar AUC or similar Cmax.
Similar AUC often results in lower Cmax, while similar Cmax results in
higher exposure in terms of AUC and Cmin. Again, knowledge of the PK-
PD relationships is needed to make these decisions.
Finally, when studying an effect of a disease state on the
pharmacokinetics, the reference group is often healthy volunteers. It should
be remembered that the phase III population might be more similar to the
test population than the reference group. A product developed for
Alzheimer’s disease showed increased exposure in patients with mild to
moderate renal impairment compared with healthy volunteers. The
magnitude of this difference was such that a dose reduction may be
considered. However, a careful look at the phase III population, where an

effective and safe dose had been established, showed that the majority of
patients had a creatinine clearance corresponding to a mild to moderate
renal impairment. In fact, the dose should possibly be increased in patients
with a relatively high filtration clearance to avoid subtherapeutic levels.
LABELING
With the aim to provide clear guidance to the prescriber, sponsors and
regulatory agencies may run a risk of simplifying the situation too much.
When deciding on a wording there may be a tendency to contraindicate the
use in a subgroup of patients when no information is available, but to
generalize too wide when limited data are provided. In the former situation,
no extrapolation from the general PK characteristics is allowed, while this is
acceptable in the latter case.
Hepatic impairment is an illustrative example of this. A drug that is
eliminated through metabolism may be contraindicated in patients with
moderate to severe impairment if no data are available (regardless of
therapeutic margin). If the sponsor provides a study with a low number of
cirrhotic patients Child-Pugh A and B, the labeling could well read “Patients
with mild to moderate hepatic impairment should be given half the
recommended maintenance dose.” This occurs despite the fact that only
cirrhotic patients were studied, that only a few were moderate according to
Child-Pugh, that there was a considerable variability in the exposure in this
group of patients, and that we know that the correlation between Child-
Pugh classification and metabolic capacity is poor. The way forward is to
accept that we sometimes cannot give clear guidance. When this is not
possible we should provide the prescriber with the information available.
This could include general pharmacokinetic characteristics of relevance for
the subgroup together with available specific information including the type
of patients in which the information was obtained (e.g., cirrhotic patients).
The prescriber can then decide what to do without being faced with a
contraindication based more on “lack of data” than a real clinical concern.
For more detailed guidance recommendations, readers are encouraged to
refer to the Guidelines on renal and hepatic impairment from FDA [26, 30]
and EU (CPMP) [27, 31].
CONCLUSIONS
It is unreasonable to require that efficacy and safety is established in phase

III studies including all subpopulations that could be treated with a new
medicinal product once on the market. To limit the size of these large

comparative studies, we must accept that measures (i.e., inclusion/

exclusion criteria) are taken to reduce the interindividual variability.
Hence, we must use other tools, such as pharmacokinetic,
pharmacodynamic, or animal studies to predict safety and efficacy in these
patients. When this is not possible or a risk is identified, the prescriber
must be informed in a proper way.
Globally, there are a number of regulatory guidelines discussing studies in
subgroups of patients. Accordingly, the marketing applications submitted to
regulatory agencies today often include studies in relevant subgroups. This
may result in specific dosing recommendations. Without that information,
regulatory agencies may have elected to contraindicate that subgroup.
Given the discussions in this chapter, the obvious question is if we are
simplifying the matter too much today? Possibly sponsors are not taking full
advantage of their scientific expertise when designing and interpreting the
results from these studies? And perhaps regulatory agencies are too willing
to contraindicate subgroups when information is not available and
extrapolate too widely when some, but perhaps insufficient information is
present?
If this is the case, it is in the interest of the patient to stimulate sponsors to
perform better scientific studies and to provide prescribers with more
precise information about available knowledge. This would put them in a
better position when deciding if and how to treat an individual patient.
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16
Clinical Pharmacology Issues Related
to Specific Drug Classes During
Drug Development
Kellie Schoolar Reynolds,*

Vanitha J.Sekar, and
Suresh Doddapaneni
INTRODUCTION
Clinical pharmacology plays a role throughout the development process of

drugs in all therapeutic classes. Three conferences convened during the
1990s addressed the utility of clinical pharmacology information in the drug
development process. The first conference, “The Integration of
Pharmacokinetic, Pharmacodynamic, and Toxicokinetic Principles in
Rational Drug Development,” occurred in 1991 in Arlington Virginia. The
other meetings were held in 1998: “AAPS, ACCP, ASCPT, FDA Symposium
on Clinical Pharmacology: Optimizing the Science of Drug Development”
in Arlington, Virginia, and “5th EUFEPS Conference on Optimizing Drug
Development: Fast Tracking into Human,” in Wiesbaden, Germany. The
* Current affiliation: Global Biopharmaceutics, Drug Metabolism and Pharmacokinetics,

Aventis Pharmaceuticals, Bridgewater, New Jersey.
373
374 Reynolds et al.
conference report for the 1991 meeting indicates that the coordinated
application of pharmacokinetics and pharmacodynamics provides a
rationale approach to efficient and informative drug development [1]. The
report for the two 1998 conferences states that there are a number of
opportunities for the use of clinical pharmacology principles at every step of
the drug development process. Appropriate use of clinical pharmacology
information allows one to identify and develop the best drugs with low risk
potential and also to identify failures faster [2]. Earlier chapters in this book
(Chapters 1, 2, and 4) elaborate on the utility of clinical pharmacology in
drug development.
The basic clinical pharmacology issues are similar across drug classes and
therapeutic indications. The ultimate goals are to understand the
relationship between exposure and response and to determine factors that
may alter exposure and response. Chapters 11, 12, 13, 15, and 16 describe
in detail how one achieves these goals. As a summary, the following four
steps describe the process.
Step 1: Determine desired efficacy endpoint
Step 2: Determine the relationship between exposure and response
Step 3: Determine dosing regimens that achieve the target
concentration range
Step 4: Determine factors that alter drug concentrations
The steps outlined above allow one to identify a dosing regimen to evaluate
for safety and efficacy and determine whether there are subpopulations that
need different doses. In addition, there are several other situations where it
is useful to understand the relationship between exposure and response for a
particular drug. These situations include: the development of new
formulations that are not bioequivalent to the approved formulation;
changing a dosing regimen to allow for less frequent dosing; determining
appropriate dose adjustments due to drug interactions; and extrapolating
drug efficacy and safety data from adults to pediatric patients.
The following sections describe specific clinical pharmacology and
exposure-response considerations for a number of drug classes. In all cases
the goals are the same—to understand the relationship between exposure
and response and determine factors that may alter exposure and response.
However, depending on disease and drug characteristics, the utility of the
information and the specific situations in which the information is used may
differ. Also, the initial source of information that contributes to the
exposure-response evaluation differs by drug class. In some cases there are
good animal and in vitro models, in other cases only human data are useful.
For some indications, studies in healthy volunteers provide information
about drug activity, while other indications require patients for all efficacy
studies.

Specific Drug Classes During Drug Development 375
This chapter includes two groups of narratives on clinical pharmacology

issues related to specific drug classes and indications. The first group of
narratives includes detailed descriptions of clinical pharmacology issues,
including exposure-response examples, for the following drug classes and
indications:
Human immunodeficiency virus (HIV) infection

Antibiotics
Stroke and cerebrovascular diseases
Migraine
Gastric acid related disorders
The second group of narratives includes short descriptions of special issues
for several drug classes and indications:
Neuromuscular blocking agents

Cancer chemoprevention
Antihypertensive agents
Inhalation drugs for pulmonary indications
Acute pain—the dental pain model
Immunosuppressive agents
Opioid analgesic agents
Lipid lowering agents
This chapter does not provide a prescriptive description of how to use

clinical pharmacology to develop drugs in specific drug classes. Also, there
are numerous indications and drug classes not covered in this chapter.
However, the selected narratives provide a broad range of examples of
important clinical pharmacology issues for specific drug classes. The issues
covered in this chapter can be extrapolated to other drug classes and
indications, as discussed in the chapter conclusion.
DETAILED DESCRIPTIONS OF CLINICAL PHARMACOLOGY

ISSUES FOR SPECIFIC DRUG CLASSES
Human Immunodeficiency Virus (HIV) Infection

The clinical course of HIV infection includes primary infection (acute
antiretroviral syndrome), asymptomatic infection, early symptomatic
infection, and advanced immunodeficiency with opportunistic
complications [3]. HIV RNA and CD4+ cell count are two laboratory tests
that indicate the clinical status of a patient. The measurement of HIV RNA
in plasma, also called viral load or viremia, indicates the amount of virus
circulating in the patient’s plasma. The number of CD4+ lymphocytes

376 Reynolds et al.
(CD4+ cell count) reflects patient immune status. As the viral load increases
and CD4+ cell count decreases, the risk of opportunistic infections,
malignancies, wasting, neurologic complications, and death increases [4]. In
July 1997, the Antiviral Drug Products Advisory Committee concurred that
favorable treatment-induced changes in HIV RNA levels are highly
predictive of meaningful clinical benefit and that HIV RNA measurements
may serve as endpoints in trials supporting accelerated and traditional
approvals. In addition, changes in CD4+ cell counts should be consistent
with observed HIV RNA changes [5].
The complexity of treating HIV leads to many situations where exposure-
response information is useful. Most patients take three or more
antiretroviral drugs per day, in addition to drugs that treat or prevent
opportunistic infections and treat complications of the antiretroviral agents,
so there is the potential for many drug interactions. Many of the drugs are
administered two or three times per day; some drugs have stringent food
restrictions. Exposure-response information helps determine appropriate
dose and regimen adjustments when drugs interact with each other and
when food alters exposure. Due to the large pill burden, drug companies
want to use exposure-response information to support changes in
formulations and dosing regimens. For example, a drug company may want
to change a dosing regimen from three times per day to two times per day.
When making such a change for a drug with dose-proportional
pharmacokinetics, the twice daily regimen will provide similar total
exposure to the drug (area under the concentration vs. time curve [AUC]
over 24 hours) as the three times daily regimen, but trough concentration
(concentration at the end of a dosing interval) will be lower and Cmax
(maximum concentration) will be higher. If adequate exposure-response
data are available, the drug company may use it to provide evidence that the
lower trough concentration will not compromise efficacy and the higher
Cmax will not cause unacceptable toxicity.
Various investigators have evaluated exposure-response relationships for
different classes of antiretroviral agents. A majority of the evaluations focus
on the first three approved classes of drugs—nucleoside reverse
transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase
inhibitors (NNRTIs), and protease inhibitors (PIs).
NRTIs inhibit viral replication by interfering with the DNA polymerase
function of viral reverse transcriptase. After uptake by host cells, nucleoside
analogues are converted to their active triphosphate forms by cellular
kinases [6]. The population pharmacokinetics and pharmacodynamics of
abacavir, an NRTI, were investigated in 41 HIV-1 infected antiretroviral
naïve adults [7]. Patients received blinded monotherapy with abacavir at
100, 300, or 600 mg twice daily for up to 12 weeks. The efficacy measures
used in the analysis were time-averaged changes in HIV-1 RNA and CD4+

cell count. The investigators used standard Emax and sigmoid Emax models
to evaluate exposure-response relationships for patients who completed 12
weeks of mono therapy (n=21).
The exposure-response evaluations indicated changes from baseline
values in both time-averaged HIV-1 RNA level and CD4+ cell count were
associated with abacavir AUC0-∞. There was also a relationship between the
efficacy parameters and abacavir Cmax, but the relationship was not as
strong as that with AUG. The EC50 value for the time-averaged change in
HIV-RNA level was greater than that for the CD4+ cell count, indicating
early saturation of the CD4+ cell count change. There was a modest increase
in HIV-1 RNA suppression, but no increase in the CD4+cell count, observed
at 600 mg twice daily relative to 300 mg twice daily as monotherapy. The
results from this evaluation supported the further evaluation of 300 mg
twice daily for the treatment of HIV infection.
The protease inhibitors (PIs) are associated with dramatic improvements
in immune function and decreases in viral load. Inhibition of the protease
enzyme results in the release of noninfectious and immature viral particles
[8]. The relationship between plasma indinavir concentrations and changes
in HIV RNA was evaluated in 23 protease inhibitor naïve patients [9].
Patients received indinavir 800 mg three times daily, in combination with
NRTIs. There was significant interpatient variability in indinavir AUC8,
values ranging from 5.4 to 52.3 µM*hr. As indicated in Table 1, median
AUC 8 and trough concentrations (C 8) were higher in patients with
undetectable HIV RNA (<500 copies/mL) compared to those patients with
detectable HIV RNA. However, there is a great deal of overlap in values
between the two groups.
These results indicate that variability in plasma drug concentrations
contributes to the variability in response. Thus, drug interactions or dosing
regimen changes that lead to lower indinavir concentrations may have a
negative impact on efficacy. In spite of this observation, the investigators did
not determine a threshold indinavir concentration necessary for efficacy.
Also, there is much variability in drug response that plasma drug
concentrations do not explain.
TABLE 1 Median (range) Indinavir Exposure Measure in Patients with

Detectable and Undetectable Plasma HIV RNA

378 Reynolds et al.
For simplicity, the examples above focus on information about the

relationship between exposure and efficacy. However, safety is an important
response measure. Safety concerns with NRTIs include anemia, pancreatitis,
peripheral neuropathy, and lactic acidosis. Certain NNRTIs are associated
with rash, liver toxicity, and CNS side effects. PIs contribute to
hypertriglyceridemia, hyperlipidemia, fat redistribution, and diabetes.
Many antiretroviral agents cause gastrointestinal adverse events. Adding
exposure-response evaluations for adverse events to the assessment for
efficacy adds a layer of complexity.
Although numerous investigators have evaluated relationships between
exposure and response for antiretroviral agents, there is no definitive
conclusion regarding the specific exposure measures that correlate with
efficacy or safety for each drug class. Based on the scientific principle that
maintaining plasma concentrations above a threshold necessary to inhibit
viral replication (e.g., in vitro IC50 or IC90—concentration of a drug that
inhibits viral replication by 50 or 90%, respectively) throughout an entire
dosing interval is essential, many investigators believe that the minimum
plasma concentration (Cmin) is the most important exposure measure for
predicting success with PIs and NNRTIs. This concept is based on
knowledge about the viral kinetics of HIV, which predict that suboptimal
concentrations of antiretroviral drugs result in the production of large
numbers of virions under conditions of high selective pressure. This
situation may put patients at risk of eventual virologic failure due to the
emergence of mutant HIV strains. Although the concept that Cmin is the most
important pharmacokinetic parameter is highly plausible, clinical data have
not confirmed it. For NRTIs, it is important to consider moieties other than
parent drug, because the intracellular triphosphate form of the drug is
active.
One limitation that complicates the evaluation of exposure-response for
antiviral agents is the fact that antiviral efficacy may change over time. This
change may occur because the virus develops resistance to the various drugs;
thus, the effective concentration may increase over time. This factor makes
it difficult to draw definitive conclusions from short-term studies. Concerns
about changes in viral susceptibility also lead one to consider whether
relationships determined for patients who are antiretroviral naïve will be the
same as relationships developed for patients who have received a lot of prior
therapy and may have higher baseline drug resistance.
Combination antiretroviral therapy that includes a PI is associated with
dramatic improvements in immune function and decreases in viral load.
However, there are a number of factors that limit the success of therapy with
PIs. Some of the factors include high first-pass metabolism by CYP3A,
efflux by P-glycoprotein (Pgp), difficult regimens, and drug interactions.
Many of the factors increase pharmacokinetic variability or limit

bioavailability, leading to a number of patients with suboptimal plasma

drug concentrations. The presence of suboptimal drug concentrations
increases the likelihood of drug resistance and treatment failure. To decrease
the occurrence of PI-resistance, investigators are attempting to increase
trough PI plasma concentrations. The PIs are metabolized by CYP3A.
Although all PIs inhibit CYP3A to some degree, ritonavir is a very potent
CYP3A inhibitor. Many investigators coadminister a subtherapeutic dose of
ritonavir with other PIs, to increase the concentrations of the PI. This
practice is called pharmacologic enhancement. The advantages of this
approach include raising trough drug concentrations, decreasing
interpatient variability, prolonging drug elimination half-life to allow less
frequent dosing, and decreasing pill burden. The approved product Kaletra™
is a fixed combination of the PI lopinavir with a subtherapeutic dose of
ritonavir. Lopinavir is potent HIV PI, with very low bioavailability due to
CYP3A first-pass metabolism. Adding a small dose of ritonavir increases
lopinavir plasma concentrations manyfold [10].
As the practice of PI pharmacologic enhancement continues, the
challenge is selecting the appropriate dose and regimen of the PI and
ritonavir. Different combinations lead to different changes in PI plasma
AUC, C max , and trough concentration. Most investigators try to
maximize trough and minimize Cmax, under the assumption that trough is
associated with efficacy and C max is associated with toxicity. An
improved understanding of exposure-response relationships for specific
drugs and drug classes will help in the selection of appropriate enhanced
regimens.
Antibiotics
Antibiotics are used to treat a wide range of bacterial infections, ranging

from otitis media and urinary tract infections to serious lower respiratory
tract infections and bacteremia. The primary goal of treatment with an
antibiotic is selection of a drug and dosing regimen that is active against the
infecting micro-organism at the site of action. Thus, in addition to being
active against the micro-organism, the drug and dosing regimen must
provide adequate amounts of active drug for an adequate amount of time at
the site of infection. There are a number of in vitro methods that allow one
to determine concentrations of drug that should be effective. These
sensitivity tests indicate the minimum inhibitory concentrations (MIC) and
minimum bactericidal concentration (MBC) for drug-organism pairs.
Patient immune defense system is also an important factor. If an antibiotic
inhibits the growth of an organism, but does not kill it, the patient’s immune

380 Reynolds et al.
system must be able to eradicate the micro-organism in order to achieve a

cure [11].
Information about the exposure-response relationship for many classes
of antibiotics arose due to integration of in vitro sensitivity tests, in vivo
measures of antibiotic efficacy and understanding of bacterial action and
antimicrobial action [12]. As first described by Shah et al. [13], there are two
groups of antibacterial drugs, based on their pattern of bactericidal activity.
The first group of drugs exhibit concentration-dependent killing, where
higher drug concentrations lead to a greater rate and extent of bactericidal
activity. Drugs in this group include fluoroquinolones and aminoglycosides
[14–16]. For these drugs, the ratio of plasma AUC to MIC (AUC/MIC) or
plasma Cmax to MIC (Cmax/MIC) correlates with efficacy. For the second
group of drugs, the rate and extent of bacterial kill depends on duration of
exposure and the effect saturates at low multiples of the MIC. Drugs in this
group include ß-lactams, vancomycin, clindamcin, and macrolides [13–16].
The parameter often used to predict efficacy for this group of drugs is the
time above the MIC. Complicating these two patterns of bacterial killing is
the postantibiotic effect (PAE), which is the time it takes an organism to
recover from the effects of exposure to an antibiotic. The PAE is
demonstrated in vitro by observing bacterial growth kinetics after removing
the drug [17]. However, the length of an in vitro PAE does not predict the
duration of the in vivo PAE [18, 19].
Knowledge of the general relationships discussed above is useful when
determining appropriate doses to study in infected patients. One can select
the dose for further study based on in vitro sensitivity data and
pharmacokinetic data from uninfected volunteers. Such a practice allows
dose selection to occur without exposing infected patients to suboptimal
antibiotic concentrations that may encourage the growth of resistant
organisms.
Preston et al. [20] used exposure-response information to help determine
the appropriate dose of levofloxacin for Phase III trials. The specific
objective of the study was to prospectively quantitate the relationship
between levofloxacin plasma concentrations and successful clinical and
microbiological outcomes and occurrence of adverse events. The study
included 313 patients with bacterial infections of the respiratory tract, skin,
or urinary tract. The levofloxacin dose and treatment duration varied,
depending on the site of infection. Patients received at least three
intravenous levofloxacin doses and then completed therapy with oral
levofloxacin, if medically appropriate. The primary analysis for this study
included the 134 patients with concentration-time data and an identified
organism with a determined MIC.
The clinical and microbiological response rates were 95 and 96%,
respectively. The investigators evaluated the relationship between a number

of factors and response rates, using logistic regression. The factors in the
analysis included organism, site of infection, MIC of the organism, and the
derived pharmacokinetic parameters peak, trough, AUC, Peak/MIC, AUC/
MIC, and Time>MIC. The final model for clinical outcome included Peak/
MIC ratio and site of infection as the predictors of clinical success. The
Peak/MIC ratio break point was 12.2. The clinical success rate for patients
achieving a ratio of greater than 12.2 was 99.0%; the rate for patients with
a ratio of 12.2 or less was 83.3%. The final model for microbiologic
outcome included Peak/MIC ratio as the predictor of microbiologic success.
The Peak/MIC ratio break point was 12.2. The microbiological success rates
for patients achieving a ratio of greater than 12.2 was 100%; the rate for
patients with a ratio of 12.2 or less was 80.8%. Although Peak/MIC ratio
was the most important derived pharmacokinetic measure for success,
AUC/MIC ratio also predicted clinical and microbiologic success. Peak/
MIC ratio and AUC/MIC ratio had similar predictive power because the
two parameters are highly correlated with one another.
Based on the results of this study, knowledge of patient factors that affect
pharmacokinetics, and MIC information, it is possible to select a
levofloxacin dose that offers a high probability of successful treatment. The
high success rate in this study indicates that the doses used were selected
based on a great deal of prior knowledge about the drug. However, the
study does allow greater confidence for the doses used in Phase III studies.
Drusano et al. [21] demonstrated a method for selecting a Phase II/III
dose of an antibiotic using human pharmacokinetic data and animal
pharmacodynamic data. The test agent was evernimicin, the first member of
a new class of oligosaccharide antibiotics active against gram positive
organisms. The investigators proposed that rational dose-selection decisions
can be made based on a mathematical model that uses four data sets: the
distribution of MICs for relevant clinical isolates, the distribution of the
pharmacokinetic parameter values in the population, the derived
pharmacokinetic/pharmacodynamic (PK/PD) target developed from animal
models of infection, and protein-binding characteristics of the test drug. The
animal model used was a neutropenic murine thigh infection model. Based
on the animal model, AUC/MIC was the best predictor of microbiologic
efficacy. The investigators used Monte Carlo simulations to determine the
probability of attaining the target AUC/MIC with two different evernimicin
doses and three different organisms. These investigators thus demonstrate
one way to determine antibiotic doses for clinical study, prior to exposing
large numbers of infected humans to the drug.
Both of the above examples indicate that exposure-response evaluations
can assist in the determination of appropriate antibiotic doses for study in
infected patients. In some cases the methods involve complex mathematical
manipulations. The doses selected by these methods require confirmation in

382 Reynolds et al.
clinical efficacy and safety studies. Although the methods are complex and
require further confirmation, they are of particular value in settings where
suboptimal drug concentrations may lead to bacterial resistance. Also,
the methods may decrease the time needed to identify a safe and effective
dose.
Stroke and Cerebrovascular Diseases
Stroke and associated cerebrovascular disorders are a major public health

concern and a leading cause of death in the United States and other
countries [22]. Stroke is indicated by an abrupt manifestation of neurologic
deficits secondary to an ischemic or hemorrhagic insult to a region of the
brain. There are various candidate drugs for acute stroke, such as
antithrombotic agents, anticoagulants, thrombolytic agents, and
neuroprotectants. Thrombolytic agents, such as tissue plasminogen
activator and streptokinase, are used in the management of thrombotic or
nonhemorrhagic strokes. Neuroprotective drugs are designed to limit tissue
damage and injury in the case of an infarct or hemorrhagic stroke.
Because stroke is a major cause of mortality and morbidity, much effort
focuses on the development of drugs to limit brain damage. Approaches to
the design of stroke trials and development of drugs for stroke benefit from
the use of clinical pharmacology principles, such as appropriate dose
selection, robust study designs, control of confounding factors, and
selection of optimal endpoints. The application of clinical pharmacology
principles helps provide therapeutic agents with better benefit-risk ratios
and helps identify failures as early as possible [23].
Use of appropriate preclinical animal models for stroke is important in
order to obtain early information regarding the pharmacological activity of
the drug. The appropriate use of exposure-response relationships in
preclinical drug development helps provide information that may be
difficult to obtain in human subjects. For example, the neuroprotective
effect of a novel, high-affinity serotonin (5-HT1A) agonist, BAY X3702,
was tested in a rat model of acute subdural hematoma (ASDH) using
different doses of the drug. The ischemic brain damage at four hours after
ASDH was assessed for each dose group and was significantly smaller for
the drugtreated group compared to the placebo-treated ASDH group. The
results from this preclinical model gave a preliminary indication that this
novel, high-affinity 5-HT1A agonist may have neuroprotective properties
[24].
The importance of clinical pharmacology in preclinical development of
drugs for this indication is further illustrated using an example of an
antithrombotic agent that is a selective inhibitor of Factor Xa [25]. The

progression of this candidate drug to Phase I studies was facilitated by useful

PK/PD information obtained in preclinical studies. A thrombosis model in
the dog was used to establish a PK/PD relationship for this drug; the
biomarker was time to artery occlusion. Based on this study, an IC70 value of
250 ng/mL was estimated for the dog. In addition, in vitro data suggested
that the pharmacodynamic response in humans was 2.5 times more
sensitive than the response in the dog; therefore, the predicted IC70 in
humans was 100 ng/mL. This information, in combination with that
obtained from allometric scaling methods (to obtain estimates of
pharmacokinetic parameters in humans), was used to select doses for the
first Phase I study by targeting steady-state concentrations in the range of
100 ng/mL. This example emphasizes the importance of the appropriate use
of exposure-response assessments in preclinical stages of drug development,
because this can help develop rational dose selection in first-time-in-man
studies.
Historically, Phase I studies conducted in healthy volunteers provide
early information related to the safety, tolerability, and pharmacokinetics of
a drug candidate. However, Phase I studies of drugs to treat stroke can also
provide useful pharmacodynamic data to address proof of therapeutic
concept. This type of information can generally be obtained fairly quickly
and effectively in healthy volunteers. For example, for an antiplatelet agent,
RGD 891 [25] information derived from exposure-response relationships
established in Phase I studies was used to simulate optimal dosing regimens
for Phase II studies. The pharmacodynamic response (% inhibition of
platelet aggregation) observed in the actual Phase II studies in patients was
similar to that observed in the Phase I studies. Exposure-response
relationships established in the Phase I setting must be confirmed and
further explored in Phase II studies in patients. An exposure-response
database such as the one built for this antiplatelet agent can guide the design
and dosing regimens for larger Phase III studies. Although, there may be
some problems extrapolating from healthy volunteers to stroke patients,
this approach is less problematic than extrapolating from experimental
animal or in vitro studies.
Migraine
Migraine is one of the most common incapacitating headaches, and it

afflicts approximately 23 million adults in the United States, with a 15%
prevalence rate [26]. Most migraine patients suffer between one and six
attacks a month and the duration of pain for each attack lasts between 4 and
72 hours. Care of migraine patients includes terminating migraine
headache, preventing attacks, and improving quality of life. Some of the

384 Reynolds et al.
preventive agents used include beta-adrenergic blockers, calcium channel

blockers, tricyclic antidepressants, anticonvulsant medications, and
serotonin antagonists. Effective agents for treatment of acute migraine
attacks include simple or combination analgesics, nonsteroidal anti-
inflammatory drugs, ergot derivatives, selective serotonin agonists, and
antiemetics. Most existing treatments are about 50–70% effective at two
hours after administration of the drug. The placebo response is about 20–
35% [26, 27].
The most recent approach to treatment of migraine headaches is the use
of potent serotonin 5-HT1B/1D receptor agonists, which are collectively
classified as triptans. Triptans are believed to exert their action by binding to
serotonin receptors in the brain, where they induce vasoconstriction of
extracerebral blood vessels and also reduce neurogenic inflammation.
Sumatriptan was the first of these compounds developed that offered
considerably improved efficacy and tolerability over the ergot-alkaloids. At
present there are at least three other triptans available on the market that
have similar or improved pharmacokinetic properties or efficacy and
tolerability profiles compared to sumatriptin [28].
New approaches to trial design include using modeling and simulation
strategies to address trial design questions. For example, during the
development of a new triptan, the amount of useful information about the
drug class, the disease, and the patient population is high. Information from
preclinical animal models and mechanism of action are also available.
Because the amount of information available in this case is large, few
assumptions are needed to construct the models needed for trial simulations,
and the uncertainty in the model predictions is usually low. Thus, the use of
computer-assisted trial designs can help shorten and focus the development
of the antimigraine triptan.
For the development of a new triptan, the objectives of modeling and
simulations include the selection of the appropriate dose for further
development. In the example discussed below, data from a Phase II dose-
ranging study were used to develop a dose-response model for the triptan
under development. The efficacy assessment measured headache severity on
a 4-point scale (0=None, 1=Mild, 2=Moderate, 3=Severe). The
pharmacodynamic endpoint used was the percent of patients who
experienced headache relief (score of 0 or 1 at two hours).
A logistic regression model for pain relief was used to model the pain
relief data and to construct a dose-response model for the triptan under
consideration [29]. For this example, the modeling exercise was helpful in
determining the median dose to achieve a target (for example 70%) pain
relief, identifying two appropriate doses for further study (assuming that the
tolerability profile at both doses was favorable) and determining the
placebo response was approximately 40%.

Information obtained from this type of modeling effort may be put to

further use in simulations of the larger, conclusive Phase III trials prior to
actually conducting the trial. Because simulations reflect uncertainty in
model parameter values, they are useful in evaluating the distribution of
model-predicted dose-response relationships. Simulations also allow
calculation of the power to detect difference from placebo as a function of
dose and sample size.
Gastric Acid-Related Disorders
Gastric acid-related disorders include heartburn, gastric and duodenal

ulcers, symptomatic gastroesophageal reflux disease (GERD), erosive
esophagitis, and pathological hypersecretory conditions such as Zollinger-
Ellison syndrome. The conventional treatment for these acid-related
disorders is the suppression of gastric acid secretion by H2 blockers and
proton pump inhibitors (PPIs). PPIs are currently the drugs of choice in the
management of acid-related disorders. The use of antisecretory agents in
combination with antibiotics is beneficial in the healing of H-pylori related
peptic ulcers. The approved H2 blockers in the United States include
cimetidine, ranitidine, famotidine, and nizatidine. Approved PPIs include
omeprazole, esomeprazole (enantiomer of omeprazole), pantoprazole,
lansoprazole, and rabeprazole.
H2 blockers principally act via competitive inhibition of H2 receptors
located on the parietal cells of the stomach. PPIs suppress gastric acid
secretion by irreversibly inhibiting the gastric H+/K+ ATPase enzyme system
at the secretory surface of the gastric parietal cell, thus blocking the final
step of acid production. Both H2 blockers and PPIs cause dose-related
suppression of basal gastric acid secretion. However, the two classes of
drugs differ markedly in their pharmacodynamic profiles. The antisecretory
effect of H2 blockers has a rapid onset and a relatively short duration. On
the other hand, although PPIs generally have short plasma elimination
halflives of about 1–2 hours, the antisecretory effect lasts for up to 3–5 days
after drug administration [30]. The prolonged effect of PPIs is attributed to
their mechanism of action, which involves irreversible inhibition of the
proton pump. The rate-limiting step in the antisecretory action of PPIs is the
turnover of the proton pump, which is reported to have a half-life of about
50 hours.
Studies in healthy volunteers can provide a preliminary evaluation of the
potential efficacy of antisecretory agents and also dose-response
information. Administration of pentagastrin or peptone meal provides acid
stimulation in these studies. Thus, early Phase I studies designed to
characterize the pharmacokinetics of the drug product can evaluate the

386 Reynolds et al.
pharmacodynamic effect as well. Pharmacodynamic biomarkers such as

median 24-hour pH, % time gastric pH >3, and % time gastric pH >4 are
common efficacy biomarkers for antisecretory agents. Use of these
biomarkers has arisen from studies that utilized meta-analyses to determine
the degree and duration of acid inhibition necessary for optimal healing of
various acid-related disorders. The findings suggest that gastric pH has to
be elevated above 3.0 for about 16–18 hours a day for treatment of
duodenal ulcer, while gastric pH needs to be elevated above 4.0 for 16–18
hours a day for treatment of esophagitis. However, the clinical relevance
of the above biomarkers is not established. Thus, if favorable data are
obtained in healthy volunteers, then similar studies are carried out in
patients to further characterize the gastric acid antisecretory effect.
Subsequently, full clinical efficacy and safety studies can be initiated with
clinical endpoints as the outcome (e.g., % of patients healed in active
duodenal ulcer trial).
There is extensive literature that describes exposure-response
relationships for H2 blockers. In general, a direct correlation appears to exist
between plasma concentrations of H2 blockers and the acid inhibitory
activity, which may be attributed to the competitive nature of drug-receptor
interaction associated with H2 blockers [31]. Exposure-response analyses
relying on the sigmoid Emax model have been successful in predicting the time
course of acid inhibitory activity for H2 blockers [32].
Apparent exposure-response relationships are reported for most PPIs
[31–35]. Katashima et al. [33] analyzed the relationship between plasma
concentrations and the inhibitory effects of the PPIs omeprazole,
lansoprazole, and pantoprazole on gastric acid secretion in healthy human
subjects using a model that assumed a linear relationship between the
fraction of inactive gastric proton pumps and the acid inhibitory effect. The
authors concluded that the potency of the acid inhibitory activity of
pantoprazole was weaker than that of omeprazole and lansoprazole, but the
apparent recovery half-life of pantoprazole (45.9 hours) was slower than
that of either omeprazole (27.5 hours) or lansoprazole (12.9 hours). It is
noteworthy that while the model reasonably predicted the gastric acid
inhibitory effects of studied PPIs, it may not have an actual mechanistic
basis. More recently, Perron et al. [34] analyzed the exposure-response
relationship for pantoprazole (10–80mg, IV & oral) in healthy human
subjects using an indirect response model. The model reasonably described
the time course of acid secretion at all studied doses. The authors concluded
that maximum acid inhibition was related to the extent of exposure to
pantoprazole. In addition, the time to maximum acid inhibition decreased
with higher doses. Further work is needed in the area of exposure-response
modeling of PPIs to fully characterize the time course of gastric acid

inhibition exerted by PPIs. More importantly, further investigation is needed

to explore the nature of the relationship between gastric acid inhibition and
clinical efficacy in acid-related gastrointestinal disorders.
Pharmacodynamic data on antisecretory activity are useful in special
populations and other situations in which clinical efficacy trials are not
feasible. For example, measurement of antisecretory activity in pediatric
patients is feasible and can be used in lieu of large clinical studies with
efficacy endpoints. Pharmacodynamic data on antisecretory activity can
also be obtained in special populations such as hepatic and renal
impairment patients. The need for dosage adjustment in these special
populations can be made by taking into account both pharmacokinetics and
pharmacodynamcis. For many other disease states, the need for dosage
adjustments in special populations are made based on pharmacokinetic data
alone.
Two key clinical pharmacology issues arise with antisecretory treatment.
The first issue is the potential effect of these agents on the absorption of
coadministered drugs. Because these drugs markedly elevate the pH in the
stomach, they may affect the pharmacokinetics of a coadministered drug
with pH-dependent absorption or a modified-release drug product with pH-
dependent drug release. For example, in normal subjects, coadministration
of rabeprazole 20 mg once daily resulted in an approximately 30% decrease
in the bioavailability of ketoconazole and increases in digoxin AUC and
Cmax of 19% and 29%, respectively [36]. Consequently, one may need to
alter the time of drug administration or adjust the dose of the
coadministered drug. The second issue is the effect of CYP2C19 phenotype
on pharmacokinetics. Omeprazole, lansoprazole, pantoprazole, and
esomeprazole are metabolized by CYP2C19, an enzyme that exhibits
genetic polymorphism; approximately 3% of Caucasians and 17–23% of
Asians are poor metabolizers. One can use exposure-response information
to determine the need for dosage adjustment in these patients.
SPECIAL CLINICAL PHARMACOLOGY ISSUES FOR

SPECIFIC DRUG CLASSES
Neuromuscular Blocking Agents
Neuromuscular blocking agents are used as adjuncts to general anesthesia

to facilitate tracheal intubation and to provide skeletal muscle relaxation
during surgical procedures. Rocuronium, vecuronium, pancuronium, and
cisatricurium are some of the nondepolarizing neuromuscular blocking
agents approved in the United States. These agents act by competing for

388 Reynolds et al.
cholinergic receptors at the motor end plate. Acetylcholinesterase inhibitors

such as neostigmine, edrophonium, and pyridostigmine reverse the
neuromuscular blockade by inhibiting the acetylcholine antagonism.
The exposure-response evaluation of neuromuscular blocking agents is
aided by a quantifiable response endpoint. The response endpoint
commonly used for evaluation of neuromuscular agents is mechanical
response to train-of-four (TOF) stimulation measured at the adductor
pollicis. There is an established and accepted methodology for
administration of the stimulus to adductor pollicis and quantification of the
response. Supramaximal square-wave TOF stimuli of 0.1–0.2 milliseconds
duration are administered at 0.1–2 Hz every 12–20 seconds to the right
ulnar nerve via surface electrodes placed at the wrist. The evoked tension of
thumb adduction is measured with a calibrated transducer. Depression of
the twitch response to the first stimulation in the TOF (T1), expressed as a
percentage of the baseline value obtained prior to the administration of the
drug, is used as a measure of the neuromuscular block. The relationship
between plasma concentrations and neuromuscular block correlate
consistently using the Sigmoid Emax model.
Exposure-response relationships for neuromuscular blocking agents have
been successfully used to compare the features of a new drug relative to
other drugs, to assess the contribution of a metabolite to the activity of a
drug, and to assess the differences in special populations for potential
dosage adjustments. For example, data obtained after separate
administration of rapacuronium bromide and its 3-hydroxy metabolite
showed that the metabolite has slower onset and higher potency (smaller
EC50 value) than rapacuronium bromide [37]. Such data obtained in early
Phase I trials can aid in the selection of the compound (parent or active
metabolite) for further development. Such data can also be used to compare
a product under development with products currently in use. Finally,
evaluation of exposure-response data for cisatricurium indicated that the
onset of effect may be marginally delayed, but otherwise there are no
distinguishable differences in the effects observed in elderly patients
compared to young adult patients [38].
Cancer Chemoprevention
Cancer chemoprevention refers to the inhibition or reversal of

carcinogenesis using appropriate pharmacologically active agents to block
the development of cancers in human beings. The goals of cancer
chemoprevention are inhibition of carcinogens, logical intervention for
persons at genetic risk for cancer, treatment of precancerous lesions, and

confirmation and translation of leads from dietary epidemiology into

intervention strategies [39].
The development and the evaluation of cancer chemoprevention
strategies involve the use of a wide range of biomarkers. The term
“biomarker” refers to internal indicators of exposure (biomarkers of
exposure), indicators of adverse effect or desired effect (biomarkers of
effect) or indicators of an intrinsic or acquired susceptibility to disease
(biomarkers of susceptibility). Biomarkers help define exposure and disease
status and may help identify possible interactions between risk factors and
disease occurrence. A biomarker needs to be validated and its distribution in
large populations described before it can be used reliably in clinical research.
In chemoprevention, an exposure biomarker is a biologic substance that
reflects endogenous or exogenous exposure to carcinogenic risk factors; this
biomarker may be predictive of the incidence or outcome of disease.
Exposure biomarkers may be used for assessment of exposure to external
carcinogens such as DNA or protein adducts or for assessment of harmful
endogenous agents such as abnormal hormonal levels [40]. A biomarker of
intermediate effect is an indicator of the development of carcinogenic
change (short of invasive cancer) in a patient. Examples of intermediate-
effect biomarkers include: (1) adenomas for colorectal cancer—in
chemoprevention trials for colorectal cancer, adenomatous polyps are used
as biomarkers of risk; (2) the degree of mammary density as a proportion of
the breast is associated with increased risk of breast cancer; and (3) tests for
p53 mutations may indicate long-term changes for liver cancers [40]. A
biomarker of susceptibility is an indicator of the ability of a patient to
respond to the challenge of a carcinogenic agent. Biomarkers of
susceptibility can help select high risk patient populations. For example,
patients diagnosed with one type of cancer are at increased risk of a second
primary cancer. Individuals in families with a genetic history of cancer may
be more susceptible [40].
The development of the nonsteroidal anti-inflammatory agent sulindac
as a chemopreventive drug used exposure-response assessments based on a
biomarker, the inhibition of cyclooxeganse 2 (Cox-2), and enhancement of
apoptosis [41]. Another example is the development of the R-isomer of
flurbiprofen [42], which works in animal models as an antiproliferative
agent against colon polyps, colonocytes, and adenocarcinomas, without the
gastrointestinal toxicity of the S-isomer or the racemate. Although these
biomarkers are not validated as surrogate endpoints, they may be used
during drug development to help assess activity of compounds to prevent
cancers. However, efficacy studies are needed to confirm the utility of the
compounds.

390 Reynolds et al.
Antihypertensive Agents
Exposure-response information plays an important role in the development
of drugs for the treatment of many cardiovascular illnesses, including
hypertension. The surrogate markers measured as response for
antihypertensive drugs include changes in blood pressure. Exposure-
response data are usually collected in Phase II trials that are double-blind,
randomized, placebo-controlled, and parallel-group in design. In the
development of antihypertensive drugs such as the angiotensin-converting
enzyme (ACE) inhibitors or beta blockers, it is important that exposure-
response information be obtained across several orders of magnitude of
doses in order to be able to determine the optimum dose for patients. In
October 2000, the FDA convened an advisory committee meeting to discuss
the importance of obtaining appropriate dose-response information during
antihypertensive drug development. The committee concluded that
elucidating the full range of dose-response relationships for antihypertensive
drugs does not constitute an undue burden on investigators, and may help
avoid the conduct of trials and experiments that do not contribute to the
total knowledge of the appropriate exposure-response relationship.
Inhalation Drugs for Pulmonary Indications

Inhalation drugs used for pulmonary indications, such as asthma, present
challenging exposure-response issues. It is presumed that the site of action is
the local airway, so systemic exposure does not represent exposure at the site
of action. Thus, systemic exposure does not predict clinical efficacy or local
safety in the respiratory tract. There are some tools for assessing the extent
of delivery to the lung. One common tool is scintigraphy. Systemic exposure
is a possible tool when the drug has low oral bioavailability and high
pulmonary bioavailability. However, these methods do not offer definitive
proof of delivery to the relevant area of the lung. Thus, one must conduct a
pharmacodynamic study to determine relevant doses for further study. The
pharmacodynamic endpoints vary depending on the class of drugs. There
are a number of direct measures of action for bronchodilators, including
serial spirometry, protection against bronchoprovacation, and peak flow-
rate assessments. There are no definitive direct measures of action for
inhaled corticosteroids, but indirect measures include exacerbation rates,
rescue use, and protection against bronchoprovocation.
In addition to endpoint issues, it is important to consider drug-device-
patient interactions for orally inhaled drugs. Different devices (metered dose
inhalers, dry powder inhalers, nebulizers) provide different patterns of
deposition in the lung. Particle size also affects where the drug deposits,
from the upper airway to the lower airway, or even being exhaled without

deposition. It is also important to consider the effect of study population.

One study indicated that following oral inhalation of fluticasone
propionate, plasma concentrations were more than twice as high in normal
volunteers compared to asthmatic patients [43].
Acute Pain—The Dental Pain Model

There are a number of situations in which patients experience acute pain.
The post third molar extraction dental pain model is a useful model for the
study of analgesia of acute pain. As described by Averbuch and Katzper
[44], the model is relatively easy to study and there are few confounding
factors. The dental pain studies are conducted in subjects scheduled to have
their third molars removed. To be included in analysis, the subjects must
experience moderate to severe pain following the extraction procedure. The
study drug is administered after the pain assessment. Subjects can receive a
local anesthetic, intravenous sedative agents, or antianxiety agents during
the surgery; however, subjects cannot receive any analgesic for 24 hours
prior to study. The efficacy endpoints include pain intensity score measured
by a 4-point categorical scale (from 0=none to 3=severe) and pain relief
score measured by a 5-point categorical scale (from 0=no relief to
4=complete relief). The scores are determined beginning just prior to drug
administration and at various times until six hours postdose. Rescue
analgesia medication is allowed, but subjects are excluded from further pain
measurements afterwards. A measure of efficacy is the pain intensity
difference (PID). The PID is calculated by subtracting the pain intensity at a
specific assessment time from the baseline score. Positive values indicate a
lessening of the patient’s pain, while a negative value indicates increasing
pain. One problem pointed out by Averbuch and Katzper is that patients
who begin with severe pain can achieve a greater reduction in pain than
patients who begin with moderate pain. One can stratify patients by
baseline pain severity for statistical analysis. Investigators can use the dental
pain model to compare two or more different drugs, to compare a new dug
to placebo, or to evaluate several different doses of one drug. Although the
dental pain model is simple and well defined, it is not clear how well the
model represents all acute pain situations.
Immunosuppressive Agents
Immunosuppressive agents are used to prevent rejection of transplanted
organs. Solid organ transplant recipients usually receive at least three
antirejection agents, making it difficult to determine the contribution of a
particular agent. The endpoint for evaluating these agents is occurrence of
organ rejection. In many cases, the symptoms of rejection are similar to

392 Reynolds et al.
adverse effects of some of the drugs patients receive. Thus, it is often

necessary to confirm acute rejection with a biopsy. The factors listed above
complicate the evaluation of exposure-response relationships for
immunosuppressive agents. However, because the transplant community
appreciates the importance of exposure-response relationships for the safety
and efficacy of immunosuppressive agents, studies of these relationships are
common.
Van Gelder, et al. [45] evaluated the relationship between exposure and
response for kidney transplant recipients receiving mycophenolate mofetil
(MMF). Mycophenolate mofetil is a prodrug for the active moiety
mycophenolic acid (MPA). The investigators randomized 154 adult
recipients of kidney transplants to receive MMF treatment targeted at three
predefined MPA AUC values (16.1, 32.2, and 60.6 µg*hr/mL). During the
first six months after transplantation, investigators collected plasma
samples for nine AUC evaluations. The primary endpoint of this six-month
study was occurrence of biopsy-proven rejection. The analysis indicated
that MPA predose concentration and MPA AUC are significantly related to
the incidence of biopsy-proven rejection, and MMF dose is significantly
related to the incidence of adverse events.
Although the study described above indicates it is possible to determine
an exposure-response relationship for immunosuppressive agents, using the
information to select a dose for patients is not simple. Most of the oral
immunosuppressive agents have high inter- or intrapatient pharmacokinetic
variability. Also, because the transplanted organ may participate in
elimination of the drug, the pharmacokinetics of the drug may vary based
on the time post transplantation. The exposure-response relationship may
vary depending on doses of the other immunosuppressive agents in the
regimen. For these reasons, transplant centers use therapeutic drug
monitoring for some agents, including cyclosporine and tacrolimus.
However, there is still debate regarding the appropriate exposure measure
for therapeutic drug monitoring—minimum plasma concentration, full
AUC, or limited sampling AUC.
Opioid Analgesic Agents

Opoid analgesic agents are used for the treatment of pain. Many of the old
opioid drugs are being reformulated into novel dosage forms for better pain
control and increased convenience. Routinely, studies characterizing the
pharmacokinetics of the drug and drug product are conducted in healthy
volunteers, to allow selection of a product with desired delivery properties.
For opioids, whether new or a reformulation, Phase I studies present a
challenge because healthy volunteers may not be able to tolerate the opioid
effects, especially at high doses. Conducting these studies in patients,

although an option, is impractical. A way to get around this problem is to

provide the volunteers naltrexone blockade. Naltrexone, an opioid
antagonist, may block the opioid effects without significantly affecting the
pharmacokinetics of the drug of interest. Bashaw et al. [46] showed that the
differences in morphine bioavailability were minimal when 60 mg
controlled-release morphine sulfate was administered with and without
naltrexone pretreatment. For other opioids, it may be worthwhile to
conduct a pharmacokinetic study first with and without naltrexone
pretreatment before such an approach is routinely adopted in other
pharmacokinetic studies.
Lipid-Lowering Agents
Atorvastatin, cerivastatin, lovastatin, and simvastatin are HMG-CoA
reductase inhibitors, a class of lipid-lowering compounds that reduce
cholesterol biosynthesis. These drugs are characterized by low (5%–60%)
and variable bioavailability attributed to extensive first-pass metabolism.
Because the CYP3A enzyme mediates metabolism of all four drugs, the
potential for significant drug-drug interactions when coadministered with
CYP3A inhibitors is high. As such, appropriate metabolism, bioavailability,
and drug interaction studies need to be conducted early during the
development of a drug belonging to this class. The safety of the drug at
doses comparable to the exposures seen in drug interaction studies can then
be studied in patient populations in safety studies to make an informed
decision regarding the safety of the drug in those situations.
Conclusions
As indicated in the introduction to this chapter, for most drug classes the
goals of clinical pharmacology and exposure-response evaluations are the
same—to understand the relationship between exposure and response and
determine factors that may alter exposure and response. However, the
utility of clinical pharmacology information throughout the various stages
of drug development differs among drug classes.
The initial sources of information that contribute to the
exposureresponse evaluation differ by drug class. Prior to human studies, in
vitro studies for anti-HIV drugs and antibiotics provide estimates of target
plasma concentrations for efficacy. Animal models provide an early
evaluation of potential efficacy for some drug classes, including antibiotics
and drugs to treat stroke and migraine. Although studies in healthy
volunteers usually provide pharmacokinetic and safety information, the
studies can provide activity and efficacy information for some drug classes,
including drugs to treat stroke and gastric acid-related disorders. However,

394 Reynolds et al.
there are many drug classes that require actual patients for evaluation of
drug activity, such as anti-HIV drugs, antibiotics, and drugs to treat
migraine. Irrespective of whether efficacy and activity information can be
obtained in healthy volunteers, the true or final assessment of safety and
efficacy for any drug can only be conducted in target patient population.
A number of drug classes present clinical pharmacology challenges.
Although drug interactions are possible with many classes of drugs,
metabolism-based interactions are a particular problem with anti-HIV
drugs and HMG-CoA reductase inhibitors. Due to their effects on gastric
acid, anti-secretory agents can interact with drugs that have pH-dependent
absorption. It is difficult to determine exposure-response relationships for
inhaled drugs, because systemic concentrations are often quite low and may
not correlate with concentrations at the site of action. In situations where
patients almost always receive multiple drugs for the same indication (HIV,
organ transplantation), it is difficult to determine the contribution of
individual drugs to response. Finally, biomarkers for use in exposure-
response evaluations are not available for some drug classes.
In closing, the information in this chapter provides examples that support
the clinical pharmacology principles discussed in other chapters in this
book. Specific characteristics of the relevant disease state, patient
population, drug class, and drug product influence the utility of various
clinical pharmacology evaluations for a drug.
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17
Issues in Bioequivalence and Development of
Generic Drug Products
Barbara M.Davit and Dale P.Conner

Rockville, MD, U.S.A.
INTRODUCTION
The topic of bioequivalence evaluation of generic drug products seems

simple but stimulates intense controversy and misunderstanding. For
example, one often hears members of the public and medical experts alike
stating various opinions on the unacceptability of approved generic drug
products based on misconceptions about the determination of therapeutic
equivalence of these products to the approved reference. These
misconceptions include the belief that the Food and Drug Administration
(FDA) approves generic products that have mean differences from the
reference product of 20–25% and that generic products can differ from each
other by as much as 45%. In addition, some incorrectly assume that, since
most bioequivalence testing is carried out in normal volunteers, it does not
adequately reflect bioequivalence and therefore therapeutic equivalence in
patients. When the current bioequivalence methods and statistical criteria
399
400 Davit and Conner
are clearly understood it becomes apparent that these methods provide a

strict and robust system that provides assurance of therapeutic equivalence.
In this chapter we will discuss the rationale and methods utilized for the
demonstration of bioequivalence for regulatory purposes in the United
States. In addition, we will touch on some controversial issues and
difficulties in demonstrating bioequivalence for certain classes of drug
products.
Bioavailability is the rate and extent of drug appearance at the site of
activity. It reflects both drug substance disposition properties as well as
formulation-related effects. In contrast, bioequivalence involves the
comparison of rate and extent of drug availability between two or more
formulations containing the same drug substance. In other words,
bioequivalence is a comparison of in vivo formulation performance. At first
it might appear to be a simple matter to compare the performance of
different formulations. In most cases, for comparison of formulation
performance of systemically available drugs, the appearance of parent drug
in the blood can be effectively used to discern the rate and extent of drug
availability from different formulations. However, there are a number of
drug products for which pharmacokinetic measures in blood are not
appropriate for the demonstration of bioequivalence. These include those
drug products that are applied to the site of activity to obtain a local
therapeutic effect, i.e., the locally acting drug products. Topical products for
the treatment of skin diseases, nasal spays for the treatment of allergic
rhinitis, and inhalers for the treatment of asthma are examples of this type
of product. For any of these products, differences in product performance
cannot be adequately evaluated by attempting to measure the appearance of
the drug in blood. Often the amount of drug absorbed into the blood is very
small and difficult to measure and, more importantly, therapeutic effects are
not related to the systemic absorption of the drug.
Most studies determining bioequivalence between generic products and
the corresponding reference-listed drug products (commonly a brand-name
product approved through the new drug approval process) are based on
evaluation of blood concentration data in healthy subjects. It is true that
drug pharmacokinetic profiles may differ between healthy subjects and
particular types of patients. This is because some disease states affect
different aspects of drug substance absorption, distribution, metabolism,
and elimination. However, the effects of disease on relative formulation
performance, i.e., release of the drug substance from the drug product, are
rare. Bioequivalence studies are designed to measure and compare
formulation performance between two drug products within the same
individuals. It is expected that the relative difference in in vivo drug release
between the two formulations will be the same whether the two
formulations are tested in patients or normal subjects. Thus, generic and

Bioequivalence and Development of Generic Drug Products 401
reference-listed drug products that are bioequivalent can be substituted for

each other in patients because they will produce the same therapeutic
effect(s) and have the same safety profile. This is illustrated by findings from
a recent observational cohort study comparing effectiveness and safety in
patients switched from brand-name warfarin sodium tablets to generic
warfarin sodium tablets [1]. The generic product was approved based on
standard bioequivalence studies in normal volunteers. The observational
cohort study showed that the two products had no difference in clinical
outcome measures.
Bioequivalence studies are also submitted to the FDA in certain situations
for new drug products. For new drug products, bioequivalence
documentation can be useful to establish links between (1) early and late
clinical trial formulations; (2) formulations used in clinical trials and
stability studies, if different; (3) clinical trial formulations and the to-be-
marketed drug products; and (4) other appropriate comparisons. The same
issues of bioequivalence study design, statistical analysis, and data
interpretation apply to both new drug products and generic drug products.
FEDERAL REGULATIONS GOVERNING BIOEQUIVALENCE

STUDIES OF GENERIC DRUG PRODUCTS
Title 21 of the Code of Federal Regulations (21 CFR) Part 320 contains the
Bioavailability and Bioequivalence Requirements pertaining to registration
of generic drug products in the United States. Part 320 consists of Subpart
A, General Provisions, and Subpart B, Procedures for Determining the
Bioavailability and Bioequivalence of Drug Products. Subpart A describes
general provisions including definitions of bioavailability and
bioequivalence. Subpart B states the basis for demonstrating in vivo
bioavailability or bioequivalence and lists types of evidence to establish
bioavailability or bioequivalence, in descending order of accuracy,
sensitivity, and reproducibility. Subpart B also provides guidelines for the
conduct and design of an in vivo bioavailability study and lists criteria for
waiving evidence of in vivo bioequivalence (bio waivers). The bio waiver
regulations apply to all parenteral solutions, including intraocular,
intravenous, subcutaneous, intramuscular, intraarterial, intrathecal,
intrasternal, and interperitoneal, but do not permit automatic waivers for all
topical and nonsystemically absorbed oral dosage products [2]. In addition,
biowaivers can be granted for ophthalmic, otic, topical, and oral solutions.
Finally, biowaivers can be granted for a number of oral drug products
approved before 1962 and formally evaluated in the late 1960s by a
Congressionally mandated panel of scientific experts under the drug efficacy
study implementation (DESI). The DESI panel formulated a list of pre-1962

drugs that had demonstrated effectiveness and lacked bioequivalence

problems [3]. For these DESI-effective drugs, the FDA waives in vivo studies
provided that formulation and in vitro dissolution data are acceptable.
STATISTICAL EVALUATION OF BIOEQUIVALENCE DATA

Statistical evaluation of most bioequivalence studies is based on analysis of
drug serum, plasma, or whole blood concentration data. The area under the
plasma concentration vs. time curve (AUC) is used as an index of the extent
of drug absorption. Generally, both AUC determined until the last
quantifiable concentration sampled (AUC0-t) and AUC extrapolated to
infinity (AUC∞) are evaluated. Maximum postdose plasma concentration
(Cmax) is used as an index of the rate of drug absorption.
To statistically compare generic and innovator AUC and Cmax data, the
FDA uses the two one-sided tests statistical procedure, also referred to as the
90% confidence interval approach. The two one-sided tests procedure
encompasses two questions [4]. Stated simply, the first test asks if the test
(generic) product is significantly less bioavailable than the reference (usually
brand-name) product. The second question asks if the reference product is
significantly less bioavailable than the test product. A significant difference
is defined as 20% at the alpha equals 0.05 level. Based on these statistical
criteria, the mean test/reference ratio of the data is usually close to one. The
criteria above may be restated to illustrate the rationale for the 0.80-1.25 (or
80%-125%) confidence interval criteria. In the first case illustrated above,
test/reference=0.80 and in the second case (or bioequivalence limit)
reference/test=0.80 (expressed by convention as test/reference=1.25, i.e., the
reciprocal of 0.80). This may be stated in clinical terms as follows. If a
patient is currently receiving a brand-name reference product and is
switched to a generic product, the generic product should not deliver
significantly less drug to the patient than the brand-name product;
conversely, if a patient is currently receiving the generic product and is
switched to the brand-name reference product the brand-name product
should not deliver significantly less drug to the patient than the generic.
CURRENT METHODS AND CRITERIA FOR DOCUMENTING

BIOEQUIVALENCE
The FDA Guidance for Industry, Bioavailability and Bioequivalence Studies

for Orally Administered Drug Products—General Considerations, provides
recommendations to firms planning to include bioavailability and
bioequivalence information for orally administered drug products in
regulatory submissions [5]. The guidance addresses how to meet the

Bioavailability/Bioequivalence Requirements set forth in 21 CFR Part 320

as they apply to oral dosage forms. The guidance also applies to nonorally
administered drug products where reliance on systemic exposure measures
is suitable to document bioavailability/bioequivalence (e.g., transdermal
systems, certain rectal, and nasal drug products.). The guidance is applicable
to both generic products and new drug products.
There are several types of studies commonly used for demonstration of
bioequivalence. The preferred study for most orally administered dosage
forms is a two-way crossover, two-period, two-sequence single-dose study,
under fasting conditions performed in normal healthy volunteers. In this
design, each study subject receives each treatment, test and reference, in
random order. Plasma or blood samples are collected for approximately
three pharmacokinetic elimination half-lives for determination of the rate
and extent of drug release from the dosage form and absorption by each
subject. A washout period is scheduled between the two periods to allow the
subjects to completely eliminate the drug absorbed from the first dose before
administering of the second dose. Although this design is carried out for
most orally absorbed drug products, it may become impractical for drugs
with long pharmacokinetic half-lives, i.e., longer than 30 hours (e.g.,
amiodarone, clomiphene). In this case a single-dose parallel design may be
used instead [6]. For drugs with very long half-lives, concentration sampling
may be carried out for a period of time corresponding to two times the
median Tmax (time to Cmax) for the product. For drugs that demonstrate low
intrasubject variability in distribution and clearance, an AUC truncated at
72 hours may be used in place of AUC0-t or AUC4 [5]. An alternative study
design that is recommended for modified-release products and for highly
variable drug products is a replicate design [5]. In this design, each
treatment is repeated in the same subject on two separate occasions. This is
performed as either a partial (three-way) or full (four-way) replication of
treatments.
Because food can influence the bioequivalence between test and reference
products, the FDA recommends that applicants developing generic products
(ANDA applicants) for oral administration conduct bioequivalence studies
under fed conditions in addition to the fasting bioequivalence studies [7].
Fed bioequivalence studies should be conducted for all generic modified-
release oral dosage forms because the bioavailability of these products is
likely to be altered by coadministration with meals. For generic immediate-
release oral dosage forms, the FDA recommends fed bioequivalence studies
whenever the label of the reference-listed drug makes statements about the
effect of food on the bioavailability of the drug product. Fed bioequivalence
studies are not recommended for generic products if the label states that the
product should be taken only on an empty stomach. Thus, the majority of
regulatory submissions for generic drug products for oral administration

will include at least two in vivo bioequivalence studies: one under fasting
conditions and one under fed conditions.
By contrast, for new drug products, fed bioequivalence studies are rarely
conducted. As previously stated, for new drug products, bioequivalence
studies are conducted to compare to-be-marketed formulations with the
clinical trial formulations and, in some circumstances, to compare new
formulations with previously approved formulations. The FDA
recommends that such bioequivalence studies for new drug products
should be generally conducted in fasted subjects [7]. Applicants
developing new drug products for oral administration usually conduct
separate studies designed to directly compare drug bioavailability in fed and
fasted subjects.
Fed bioequivalence studies are generally conducted using meal conditions
expected to provide the greatest effects on formulation performance and
gastrointestinal physiology such that systemic drug bioavailability is
maximally effected. Typically, the drug is administered to subjects within 30
minutes of consuming a high-fat, high-calorie meal. The FDA recommends
that these studies use a randomized, balanced, single-dose, two-treatment,
two-period, two-sequence crossover design [5]. For a few drug products,
such as mefloquine, the FDA recommends that applicants evaluate
bioequivalence only under fed conditions because there are safety concerns
associated with administration of the product on an empty stomach.
The FDA recommends that in vivo bioequivalence studies be conducted
in individuals representative of the general population, taking into account
age, sex, and race factors [5]. For example, if a drug product is to be used in
both sexes, the sponsor should attempt to include similar proportions of
males and females in the study; if the drug product is to be used
predominantly in the elderly, the applicant should attempt to include as
many subjects of 60 years of age or greater as possible. Restrictions on
admission into the study should generally be based solely on safety
considerations.
Bioequivalence studies should be conducted in the intended patient
population when there are significant safety concerns associated with use in
healthy subjects. For example, an antineoplastic drug intended for short-
term therapy, such as etoposide, can be evaluated following a single dose
either in cancer patients in remission or in patients under active treatment by
sampling on the first day of a treatment cycle. As another example, for the
medication clozapine, normal subjects may experience serious orthostatic
hypotension with the first dose. Moreover, clozapine requires dose titration
to achieve the maximum-tolerated, approved regimen, which is generally
achieved using multiples of the highest approved strength. Thus, for
clozapine, the most appropriate study design is a steady-state (multiple
dose) crossover bioequivalence study in patients [8].

TYPES OF EVIDENCE TO ESTABLISH BIOAVAILABILITY

AND BIOEQUIVALENCE
General Considerations
Subpart B of the Bioavailability and Bioequivalence Requirements in 21
CFR Part 320 lists the following in vivo and in vitro approaches to
determining bioequivalence in descending order of accuracy, sensitivity, and
reproducibility [9]:
• In vivo measurement of active moiety or moieties in blood,

plasma, or serum.
• In vivo measurement of the active moiety in urine.
• In vivo pharmacologic (pharmacodynamic) comparison.
• Well-controlled clinical trials.
• In vitro comparison.
• Any other approach deemed appropriate by FDA.
Figure 1 illustrates, for a model of oral dosage form performance, why the
most sensitive approach is to measure the drug in biological fluids, such as
blood, plasma, or serum. The active ingredient leaves the solid dosage form
and dissolves in the gastrointestinal tract, and following absorption through
the gut wall, appears in the systemic circulation. The step involving
dissolution of the drug substance prior to absorption is the critical step,
necessary for the absorption of the drug, that is determined by the
formulation. Other steps illustrated in the diagram are patient- or subject-
determined processes not directly related to formulation performance.
Variability of the measured endpoint increases with each additional step in
the process. Therefore, variability of clinical measures is quite high
compared to blood concentration measures. Figure 2 shows that the blood
concentration of a drug directly reflects the amount of drug delivered from
the dosage form.
In situations where a drug cannot be reliably measured in blood, it may
be appropriate to base bioequivalence evaluation on an in vivo test in
humans in which an acute pharmacologic (pharmacodynamic) effect is
measured as a function of time. Generally, the pharmacodynamic response
plotted against the logarithm of dose appears as a sigmoidal curve, as shown
in Fig. 3. It is assumed that, after absorption from the site of delivery, the
drug or active metabolite is delivered to the site of activity and, through
binding to a receptor or some other mechanism, elicits a quantifiable
pharmacodynamic response. Since additional steps contribute to the
observed pharmacodynamic response, a pharmacodynamic assay is not as
sensitive to drug formulation performance as blood drug concentrations. In

FIGURE 1 The most sensitive approach in evaluating bioequivalence of two

formulations is to measure drug concentration in biological fluids, as illustrated in
this diagram showing the relationship between dosage form performance and
therapeutic response. Following oral dosing, the active ingredient leaves the solid
dosage form, dissolves in the gastrointestinal tract, and, following absorption
through the gut wall, appears in the systemic circulation. Formulation performance
is the major factor determining the critical steps of dosage form disintegration and
drug substance dissolution prior to absorption. All other steps following in vivo drug
substance dissolution are patient-or subject-determined processes not directly
related to formulation performance. The variability of the measured endpoint
increases with each additional step in the process, such that variability of clinical
measures is quite high compared to that of blood concentration measures. As a
result, a pharmacodynamic or clinical approach is not as accurate, sensitive, and
reproducible as an approach based on plasma concentrations.
developing a pharmacodynamic assay for bioequivalence evaluation, it is

critical to select the correct dose. The dose should be in the range that
produces a change in response, as shown in the midportion of the curve. In
other words, the pharmacodynamic assay should be sensitive to small
changes in dose. A dose that is too high will produce a minimal response at
the plateau phase of the dose-response curve, such that even large
differences in dose will show little or no change in pharmacodynamic effect.
Depending on the type of response, a pharmacodynamic study can be
conducted in healthy subjects. The pharmacodynamic response selected
should directly reflect dosage form performance and availability at the site
of activity but may not necessarily reflect therapeutic efficacy.

FIGURE 2 The blood concentration of a drug directly reflects the amount of drug
delivered from the dosage form. The corresponding responses over a wide range of
doses will be of adequate sensitivity to detect differences in bioavailability between
two formulations. This is illustrated for two widely different doses, D1 and D2. Any
differences in dosage form performance are reflected directly by changes in blood
concentration (R1 and R2).
If it is not possible to develop reliable bioanalytical or pharmacodynamic

assays, then it may be necessary to evaluate bioequivalence in a well-
controlled trial with clinical endpoints. This type of bioequivalence study is
conducted in patients and is based on evaluation of a therapeutic, i.e.,
clinical response. The clinical response follows a similar dose-response
pattern to the pharmacodynamic response, as shown in Fig. 3. Thus, in
designing bioequivalence studies with clinical endpoints, the same
considerations for dose selection apply as for bioequivalence studies with
pharmacodynamic endpoints. As with a pharmacodynamic study, the
appropriate dose for a bioequivalence study with clinical endpoints should
be on the linear rising portion of the dose-response curve, since a response in
this range will be the most sensitive to changes in formulation performance.
Due to high variability and the sometimes subjective nature of clinical
evaluations, the clinical response is often not as sensitive to differences in
drug formulation performance as a pharmacodynamic response. For these

FIGURE 3 In evaluating bioequivalence in a study with pharmacodynamic or

clinical endpoints, it is critical to select a dose that falls on the middle ascending
portion of the sigmoidal dose—response curve. The most appropriate dose for a
study based on pharmacodynamic or clinical endpoints should be in the range that
produces a change in response (R1), as shown in the midportion of the curve (D1).
A dose that is too high will produce a minimal response at the plateau phase of the
dose—response curve, such that even large differences in dose (D2) will show little
or no change in pharmacodynamic or clinical effect (R2). Thus, two formulations
which are quite different may appear to be bioequivalent.
reasons, the clinical approach is the least accurate, sensitive, and

reproducible of the in vivo approaches to determining bioequivalence.
Blood, Plasma, or Serum
Most bioequivalence studies submitted to the FDA are based on measuring

drug concentrations in plasma. In certain cases, whole blood or serum may
be more appropriate for analysis. Measurement of only the parent drug
released from the dosage form, rather than a metabolite, is generally
recommended because the concentration-time profile of the parent drug is
more sensitive to formulation performance than a metabolite, which is more
reflective of metabolite formation, distribution, and elimination [5].
Measurement of a metabolite may be preferred when parent drug

concentrations are too low to permit reliable measurement. Metabolites

formed by presystemic metabolism that contribute meaningfully to safety
and efficacy are also measured in addition to the parent.
Urine
Urine measurements are not as sensitive as plasma measurements, but are
necessary for some drugs such as orally administered potassium chloride
[10], for which serum concentrations do not accurately reflect the amount
of drug absorbed from the dosage form. Both cumulative amount of drug
excreted (Ae) and maximum rate of urinary excretion (Rmax) are evaluated
statistically in bioequivalence studies which rely on urine concentrations.
Studies of Pharmacologic (Pharmacodynamic) Effects

The FDA accepts pharmacodynamic effect methodology to approve generic
topical corticosteroid drug products [11]. This approach is based on the
ability of corticosteroids to produce blanching or vasoconstriction in the
microvasculature of the skin. Since this property is presumed to relate to the
amount of drug leaving the dosage form and entering the skin, the
vasoconstriction assay has become the means for assessing bioavailability
and bioequivalence of topical corticosteroids. In designing a bioequivalence
study based on vasoconstriction, an applicant should first conduct a pilot
study using the reference topical corticosteroid product to determine the
dose-duration that will give the half-maximal response (ED50). During the
pivotal study, the test and reference products are applied to subjects’
forearms for a dose-duration approximately equal to the ED50. If the ED50 is
estimated correctly in the pilot study, then the pivotal study will be
adequately sensitive to differences in formulation performance. For
bioequivalence analysis, 90% confidence intervals are determined for ratios
of test and reference area-under-the-effect-curve (AUEC) data; these should
fall within the range of 0.80-1.25.
Well-controlled Clinical Trials

Bioequivalence study designs with clinical endpoints are used with some
topical products that are active at the site of application, such as tretinoin
topical formulations. This approach is also used for some oral drug
products that are not systemically absorbed, such as sucralfate tablets.
Bioequivalence studies with clinical endpoints generally employ a
randomized, blinded, balanced, parallel design. Studies compare the efficacy
of the test product, innovator product, and placebo to determine if the two
products containing active ingredient are bioequivalent. The placebo is

included to assure that the two active treatments in the clinical trial actually
are being studied at a dose that is pharmacologically and clinically active.
Failure to assure that the treatments are clinically active in the trial would
show that the trial has no sensitivity to differences in formulation
performance, i.e., the response is on the flat bottom of the dose—response
curve (Fig. 3). A generic equivalent of the innovator product should be able
to demonstrate bioequivalence for selected clinical endpoint(s) that
adequately reflect drug appearance at the site(s) of activity and therefore
formulation performance. For example, for tretinoin topical cream
formulations indicated for treatment of acne vulgaris, the endpoints relate
to severity and number of lesions, whereas for sucralfate tablets, the clinical
endpoint is duodenal ulcer healing at four weeks [6]. The test and reference
clinical responses are considered bioequivalent if the 90% confidence
interval for the differences in proportions between test and reference
treatment is contained within the limits of -0.20 to 0.20.
In vitro Tests
With suitable justification, bioavailability and bioequivalence may be
established by in vitro studies alone. This approach is also suitable for some
types of locally acting products such as nasal solution aerosols/sprays,
which produce effects on nasal sites of action without relying upon systemic
exposure, and cholestyramine resins, which form nonabsorbable complexes
with bile acids in the intestine. The FDA evaluates in vitro bioequivalence of
nasal sprays and aerosols only for products with the same formulations
within the spray device as the corresponding innovator products [12].
Therefore, the in vitro performance measures assess comparative
performance of the devices used for administration. Test/reference ratios for
dose/spray content uniformity, droplet/particle size distribution, spray
pattern, and plume geometry measurements should be equivalent between
the two products. For cholestyramine resins, the in vitro measures of
bioequivalence are based on the rates of binding to bile acid salts [13]. The
90% confidence of the test/reference ratios of the equilibrium binding
constants should fall within the limits of 0.80 to 1.25.
Waivers of in vivo Bioequivalence based on in vitro Testing

Under certain circumstances, product quality bioavailability and
bioequivalence can be documented using in vitro approaches [9]. In vitro
dissolution testing to document bioequivalence for nonbioproblem DESI
drugs remains acceptable. In vitro dissolution characterization is
encouraged for all product formulations investigated, including prototype
formulations, particularly if in vivo absorption characteristics are being

defined for the different product formulations. Such efforts may enable the
establishment of an in vitro-in vivo correlation. When an in vitro-in vivo
correlation is available [2], the in vitro test can serve as an indicator of how
the product will perform in vivo.
DRUGS THAT ARE ALSO ENDOGENOUS SUBSTANCES
Bioequivalence studies of endogenous drug substances need special

considerations. This is because for these substances there are measurable
baseline concentrations in biological fluids, either because the product is
manufactured in the body, such as levothyroxine or ursodiol, or is available
from dietary sources, such as potassium chloride [14]. As previously stated,
bioequivalence studies are conducted to compare formulation performance.
With most drug products, the only source of the drug appearing in the blood
is from the dosage form. With endogenous substances, there are two or
more sources causing the substance to appear in blood. Adding complexity
are feedback processes with substances like hormones, circadian rhythms,
and influxes from the diet. Figure 4 shows that, following dosing with an
endogenous substance, both release from the dosage form and body
production contribute to blood levels.
Thus, in most cases, the FDA recommends baseline correction for
endogenous substances. Measurement of the endogenous baseline depends
on the characteristics of the endogenous substance. Often, a baseline is
determined from one to three measurements taken before the drug products
are given. Less often, sampling at regular intervals throughout the day for at
least two days prior to dosing is performed. The baseline sampling should
take place at several intervals to account for fluctuations due to circadian
rhythms. Corrections should be subject- and period-specific. One important
consideration in comparing generic and reference products is to give an
adequate dose, because the plasma concentrations have to be high enough
so that the substance can be accurately and reliably determined by the assay,
after baseline correction. The objective is to discern any differences between
a generic and reference product, without failing products that are almost
identical.
Potassium chloride presents a special case. Serum measurements cannot
be used for bioequivalence studies of potassium chloride products. Because
homeostatic mechanisms maintain potassium concentrations in biological
fluids within a narrow range, serum concentrations change minimally in
response to a bolus dose. As shown in Fig. 5, the baseline is very high
relative to any changes occurring after dosing. In fact, in pharmacokinetic
studies of postassium chloride tablets, following an 80 mEq dose, serum
potassium increases only about 5% relative to baseline [14]. Since virtually

FIGURE 4 Two or more sources contribute to blood levels of a drug that is already
present in the body as an endogenous substance. The drug that appears in the
blood and throughout the body arises from body production in addition to release
from the dosage form. With some endogenous substances, especially hormones,
there can be a feedback process such that production and storage of the
compound changes as blood or body concentrations change. When determining
bioequivalence of formulations of these types of drugs, it may be necessary to use
a baseline correction to account for the amount in blood that did not come from the
formulation.
all of ingested potassium is excreted in urine, measuring urine output of

potassium is an accurate means of comparing the potassium released from
generic vs. reference formulations. The FDA recommends that, for
potassium chloride bioequivalence studies, subjects ingest a standardized
potassium diet for an equilibration period of several days before sampling
takes place [10]. This practice helps achieve a relatively stable baseline
before dosing starts.
COMPLEX DRUG SUBSTANCES
There are many drug substances that may fit into the category of “Complex
Drug Substances.” These include many proteins, peptides, botanicals,
synthetic hormones, biotechnology products, and complex mixtures. For
most of these drugs, the most difficult problem is to demonstrate

FIGURE 5 Unlike endogenous substances such as hormones which are

synthesized by the body, endogenous potassium arises solely from dietary
sources. The body transports potassium from place to place and excretes excess
amounts primarily into the urine. Thus, a patient deficient in potassium will utilize
supplemental potassium, whereas normal volunteers ingesting adequate levels of
potassium will excrete virtually all, if any, excess. Because homeostatic
mechanisms maintain blood potassium levels within a narrow range, there is very
little change in blood levels following a potassium dose. This means that following a
dose of potassium, a high percentage of the resulting potassium blood levels is due
to the baseline that was already present before dosing. As a result, blood is not a
good site for sampling for bioequivalence studies of oral dosage forms delivering
potassium. Since most of an ingested dose is excreted in urine, bioequivalence is
documented by measuring amounts of potassium excreted in urine. Urinary data
must still be corrected for baseline, but this baseline represents a much smaller
percentage of the total excreted.
pharmaceutical equivalence, i.e., that the drug substances are actually the
same within each manufacturer’s dosage form. In many cases, current
technology is not sufficient to unequivocally characterize the drug substance
in two different manufacturer’s products or after a single manufacturer
wishes to make pre or postapproval changes in manufacturing procedures.
These challenges in drug substance characterization methods currently may
stand in the way of the approval of generic products for many of these
products containing complex drug substances.

NARROW THERAPEUTIC INDEX DRUGS
There are no additional approval requirements for generic versions of

narrow therapeutic index (NTI) drugs vs. non-NTI drugs. The FDA does
not set specific standards based on therapeutic index [5, 15]. The
bioequivalence criteria, using the 90% confidence interval approach, are
quite strict; there is no need to apply stricter criteria for NTI drugs. The
current FDA position is that any generic product may be switched with its
corresponding reference-listed drug.
SUMMARY
Current bioequivalence methods in the United States are designed to

provide assurance of therapeutic equivalence of all generic drug products to
their innovator counterparts. The sole objective of bioequivalence testing is
to measure and compare formulation performance between two or more
pharmaceutically equivalent drug products. For generic drugs to be
approved in the United States, they must be pharmaceutically equivalent
and bioequivalent to be considered therapeutically equivalent and therefore
approvable. In the United States, Part 320 of 21 CFR, the Bioavailability
and Bioequivalence Requirements, states the basis for demonstrating in vivo
bioequivalence, lists the types of evidence to establish bioequivalence (in
descending order of accuracy, sensitivity, and reproducibility), and provides
guidelines for the conduct and design of an in vivo bioavailability study.
Through the years, the U.S. FDA has published Guidances for Industry
which address how to meet the Bioavailability and Bioequivalence
Requirements set forth in 21 CFR Part 320. The FDA updates these
Guidances as the need arises to ensure that they reflect state-of-the art
scientific thinking regarding the most accurate and sensitive methods
available to demonstrate bioequivalence between two products. Consulting
with panels of experts such as Advisory Committees, participating in
meetings and workshops with Academia and Industry (both in the United
States and abroad), and inviting public comment on draft guidances are
among the mechanisms that the FDA employs to keep Guidance
development current.
The FDA’s current statistical criteria for determining acceptability of
bioequivalence studies are designed to assure that the test product is not
significantly less bioavailable than the reference (usually the innovator)
product, and that the reference product is not significantly less bioavailable
than the test product. The difference for each of these two tests cannot
exceed 20%, with the result that the test/reference ratios of the
bioequivalence measures must fall within the limits of 0.80 to 1.25. A

generic product which does not meet these criteria is not approved. The
FDA stipulates in the Bioavailability and Bioequivalence Regulations that
the most accurate, sensitive, and reproducible method for determining
bioequivalence is to measure drug concentrations in blood in a single-dose
study using human subjects. If it is not possible to accurately and
reproducibly measure drug concentrations in blood, other approaches may
be used, such as measuring an active metabolite or measuring drug in urine.
For locally acting drug products with little systemic availability,
bioequivalence may be evaluated by pharmacodynamic, clinical-endpoint,
or highly specialized in vitro studies. Because of the challenges of the
therapeutic equivalence criteria, there is not yet a mechanism for approving
generic versions of many complex drug substances such as proteins,
botanicals, and complex mixtures.
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6. Freedom of Information Staff, Food and Drug Administration, Center for Drug
Evaluation and Research, Rockville, MD. Summary Basis of Approval.
Center for Drug Evaluation and Research. Guidance for Industry: Food-Effect
Bioavailability and Fed. Bioequivalence Studies, January 30, 2003.
Center for Drug Evaluation and Research. Draft Guidance for Industry:
Clozapine Tablets in vivo Bioequivalence and in vitro Dissolution Testing,
December 29, 2003.
9. 57 Fed Regist 29354, July 1, 1992.
Potassium Chloride Modified-Release Tablets and Capsules: In vivo
Bioequivalence and in vitro Dissolution Testing, August 6, 2002.

Center for Drug Evaluation and Research. Guidance for Industry: Topical
Dermatologic Corticosteroids: In vivo Bioequivalence, March 6, 1998.
Bioavailability and Bioequivalence Studies for Nasal Aerosols and Nasal Sprays
for Local Action, April 2, 2003.
Center for Drug Evaluation and Research. Interim Guidance for Industry:
Cholestyramine Powder in vitro Bioequivalence, July 15, 1993.
14. Advisors and Consultants Staff, Food and Drug Administration, Center for
Drug Evaluation and Research, Rockville, MD. Meeting of the Advisory
Committee for Pharmaceutical Science, March 13, 2003.
15. S. Nightingale, From the Food and Drug Administration. JAMA 1998, 279,
645.

18
Regulatory Considerations for Oral
Extended Release Dosage Forms
and in vitro (Dissolution)/in vivo
(Bioavailability) Correlations
Ramana S.Uppoor and Patrick J.Marroum

INTRODUCTION
Optimizing drug therapy to patients is one of the important topics on the

minds of all health care personnel. Drug developers, prescribers, and
pharmacists would like to give the best drug to the patients, delivered in the
most optimal way, to be taken the least number of times per day with
maximized efficacy and minimal side effects. In this regard, modified-release
dosage forms have found extensive use in today’s pharmaceutical
armamentarium. Due to technological developments in the pharmaceutical
industry, advanced drug delivery systems are being developed to improve
patient compliance (by needing to take the drug less frequently) and, in several
cases, improved efficacy with reduced side effects. Modified-release dosage
forms have thus become very popular in improving patient therapy. These
dosage forms have sometimes also been developed to extend the patent life
417
418 Uppoor and Marroum
of the drug and drug product. The major goal in designing an extended
release (ER) product should be that of optimizing therapeutic effects and
safety of a drug, while at the same time improving patient convenience and
compliance through extended dosage intervals. In this chapter, we will
primarily focus on oral extended-release dosage forms, although the principles
can be applied to nonoral extended-release products as well, e.g., transdermal
systems. It is important to note that extended-release dosage forms are more
complex than immediate-release dosage forms. Generally one dosage unit of
extended-release product contains multiples of doses contained in an
immediate-release dosage unit. In addition, the release of the drug from the
extended-release product is intentionally modified. Therefore, it becomes
extremely important to understand the release characteristics of these products
as well as to evaluate how stable the release is under altered conditions in
vivo, e.g., different pH, presence of food, etc. Because of these complexities
involved in extended-release products, it is necessary to understand the
regulatory considerations in evaluating these drug products.
In this chapter, we will first provide definitions and then discuss the
regulatory considerations (in vivo and in vitro studies needed) for developing
and maintaining oral extended- release products on the market. Finally, we
will focus on in vitro/in vivo correlations to select meaningful dissolution
methods that will enable the dissolution test to be a surrogate for
bioequivalence. In this regard, we will provide several illustrations that will
help understand the regulatory considerations as well as highlight some of
the issues and pitfalls that arise in in vitro/in vivo correlations (IVIVC)
development/validation.
DEFINITIONS
For ease of understanding, it is important to define the following terms

before a substantial discussion of extended-release product development is
started.
Controlled-Release Dosage Forms

A class of pharmaceuticals or other biologically active products from which
a drug is released from the delivery system in a planned, predictable, and
slower than normal or conventional manner (e.g., Ocuserts, Depot
injectables such as Lupron depot) [1].
Modified-Release Dosage Forms

Dosage forms whose drug-release characteristics of time course and/or location
are chosen to accomplish therapeutic or convenience objectives not offered by

Regulatory Considerations 419
conventional dosage forms such as a solution or an immediate-release dosage

form. Modified-release solid oral dosage forms include both delayed (e.g.,
enteric-coated products) and extended-release drug products [2].
Extended Release
Extended-release products are formulated to make the drug available over
an extended period after ingestion. This allows a reduction in dosing
frequency compared to a drug presented as a conventional dosage form
(e.g., as a solution or an immediate-release dosage form) [2].
Delayed Release
Release of a drug at a time other than immediately following oral
administration e.g., enteric coated products [2].
Compositionally Proportional
All active and inactive ingredients are in exactly the same proportion
between different strengths (e.g., a tablet of 50-mg strength has all the
inactive ingredients, exactly half that of a tablet of 100-mg strength, and
twice that of a tablet of 25-mg strength).
Proportionally Similar
The phrase proportionally similar is defined in three ways [3]:
Definition 1 (compositionally proportional): All active and inactive
ingredients are in exactly the same proportion between different strengths
(e.g., a tablet of 50-mg strength has all the inactive ingredients, exactly half
that of a tablet of 100-mg strength, and twice that of a tablet of 25-mg
strength).
Definition 2: Active and inactive ingredients are not in exactly the same
proportion between different strengths as stated above, but the ratios of
inactive ingredients to total weight of the dosage form are within the limits
defined by the SUPAC-IR and SUPAC-MR guidances up to and including
Level II.
Definition 3: For high potency drug substances, where the amount of the
active drug substance in the dosage form is relatively low, the total weight of
the dosage form remains nearly the same for all strengths (within ±10% of
the total weight of the strength on which a biostudy was performed), the
same inactive ingredients are used for all strengths, and the change in any
strength is obtained by altering the amount of the active ingredients and one
or more of the inactive ingredients. The changes in the inactive ingredients

are within the limits defined by the SUPAC-IR and SUP AC-MR guidances
up to and including Level II.
In vitro/in vivo Correlations

A predictive mathematical model describing the relationship between an in
vitro property of an oral dosage form (usually the rate or extent of drug
dissolution or release) and a relevant in vivo response (e.g., plasma drug
concentrations or amount of drug absorbed) [4].
FDA BIOAVAILABILITY STUDY REQUIREMENTS FOR

CONTROLLED-RELEASE PRODUCTS—CODE OF FEDERAL
REGULATIONS
The general pharmacokinetic/biopharmaceutic requirements for controlled-
release formulations are set forth in 21 CFR 320.25(f) and are listed below
(see the chapter on CFR):
21 CFR 320.25(f): Controlled-Release Formulations [5]

1. The purpose of an in vivo bioavailability study involving a drug product
for which a controlled-release claim is made is to determine if all of the
following conditions are met:
i. The drug product meets the controlled-release claims made for it.
ii. The bioavailability profile established for the drug product rules
out the occurrence of any dose dumping.
iii. The drug product’s steady-state performance is equivalent to a
currently marketed noncontrolled-release or controlled-release
drug product that contains the same active drug ingredient or
therapeutic moiety and that is subject to an approved full new
drug application.
iv. The drug product’s formulation provides consistent
pharmacokinetic performance between individual dosage units.
The types of studies needed to address these aspects are described in the next
section.
2. The reference material(s) for such a bioavailability study shall be
chosen to permit an appropriate scientific evaluation of the controlled-
release claims made for the drug product. The reference material could be:
i. A solution or suspension of the active drug ingredient or

therapeutic moiety.

ii. A currently marketed noncontrolled-release drug product

containing the same active drug ingredient or therapeutic moiety
and administered according to the dosage recommendations in
its labeling.
iii. A currently marketed controlled-release drug product subject to
an approved full NDA containing the same active drug
ingredient or therapeutic moiety and administered according to
the dosage recommendations in its labeling.
iv. A reference material other than those discussed above that is
appropriate for valid scientific reasons.
Clinical Pharmacology and Biopharmaceutics Studies

For extended-release dosage forms, the general studies needed are listed
below [3, 6]. The first three studies listed are always necessary to address the
CFR requirements.
1. Single-dose fasting relative bioavailability/bioequivalence study

compared to a reference formulation
2. Steady-state relative bioavailability/bioequivalence study
compared to a reference formulation
3. Food—effect study
4. Dose-proportionality study
5. Dosage strength bioequivalence study
6. Single-dose bioequivalence study (clinical vs. market
formulations)
7. IVIVC
8. PK/PD evaluation
NEW DRUG APPLICATIONS VS. ABBREVIATED NEW DRUG

APPLICATIONS
Some important considerations in deciding whether an ER dosage form
should be filed as a new drug under a new drug application (NDA) or as a
generic under an abbreviated new drug application (ANDA) are:
• Whether this drug is a new molecular entity

• Whether this ER product is the first extended-release product for
that drug
• Whether there is any other similar ER product on the market
• Whether the sponsor intends to make claims of different efficacy
or safety profile for this ER product

In all the above cases, generally the ER product is submitted as an NDA. In

situations where there is already an immediate-release form of the drug that
is marketed, a 505(b)(2) NDA application could be submitted to the FDA
for approval. These regulations for a 505(b)(2) NDA are covered under 21
CFR 314.54. Any person seeking approval of a drug product that represents
a modification of a listed drug (e.g., a new indication or new dosage form)
and for which investigations, other than bioavailability or bioequivalence
studies, are essential to the approval of the changes may submit a 505(b)(2)
application (except for cases where the only difference between the
reference-listed drug and the test drug is that the extent of absorption is less
than the reference or if the rate of absorption is unintentionally less than the
reference). This application needs to contain only that information needed
to support the modification(s) of the listed drug. If, however, the drug is
already available as an ER product and the new sponsor is developing
another ER product with no intention of being different from the currently
marketed ER product, this will have to be submitted as an ANDA where one
could rely solely on bioequivalence studies.
GENERAL APPROACHES FOR EVALUATING EXTENDED-

RELEASE PRODUCTS
Are clinical trials always necessary for the approval of an ER product or can
we rely on pharmacokinetic data alone? This is a fundamental question in
evaluating ER products. A rational answer to this question is based on
evaluation of the pharmacokinetic properties and plasma concentration/
effect relationship of the drug. If there is a well-defined predictive
relationship between the plasma concentrations of the drug and the clinical
response (PK/PD for both safety and efficacy), it may be possible to rely on
plasma concentration data alone as a basis for approval of the extended-
release product. In the following situations, it is expected that clinical safety
and efficacy data be submitted for approval of the ER product NDA:
• When the ER product involves a drug which has not previously

been approved (in any dosage form), since there is no approved
reference product to which a bioequivalence claim could be made
• When the rate of input has an effect on the drug’s efficacy and
safety profile
• When a claim of therapeutic advantage is intended for the ER
product
• When there are safety concerns with regard to irreversible toxicity
• When there are uncertainties concerning the PK/PD relationships
of the drug

• Where there is evidence of functional (pharmacodynamic)

tolerance
• Where peak to trough differences of the immediate-release dosage
form are very large and the effect of input rate is unknown.
In vivo Studies Generally Necessary for Approval of ER NDAs

In cases where a new drug does not have adequate safety and efficacy
established for either IR or ER dosage forms, safety and efficacy trials are
required for an ER product. An example of such a case is where an ER
product is being developed as the first dosage form of a new drug without
prior approval or study of an IR product. As noted below, PK and PK/PD
approaches may alleviate the need to conduct all of the usual safety and
efficacy studies (i.e., a complete clinical trial program with two clinical efficacy
and safety trials) for an ER product when an IR product is already approved.
The general approaches for studying and evaluating ER products are
described below:
Demonstration of Safety and Efficacy Primarily based on

Clinical Trials
• In general, for drugs where the concentration-response
relationships are not established or are unknown, applications
for an ER product where an IR product already exists will require
the demonstration of the safety and efficacy of the product in the
target patient population. In these cases, the PK and
biopharmaceutics studies conducted to address the CFR
requirements (described in the previous section) while necessary
are mostly supportive and are usually for descriptive and labeling
purposes. These studies may also help in the initial-dose selection.
When a new molecular entity is developed as an ER formulation, additional

studies to characterize its clinical pharmacology and ADME characteristics
will be necessary.
Demonstration of Safety and Efficacy based on PK, PK/PD,

and Supportive Clinical Trials
The FDA “Guidance for Industry—Providing Clinical Evidence of
Effectiveness for Human Drug and Biologic Products” [7] indicates that in
certain cases, the clinical efficacy of modified-release dosage forms or
different dosage forms can be extrapolated from existing studies, without
the need for additional well-controlled clinical trials. This may be possible

because other types of data such as PK studies (BA/BE studies) and/or PK/
PD studies allow the application of known effectiveness to the new dosage
form.
• “Where blood levels and exposure are not very different, it may
be possible to conclude that a new form is effective on the basis
of PK data alone.”
• “Where blood levels are quite different, if there is a well-
understood relationship between blood concentration and
response, including an understanding of the time course of that
relationship, it may be possible to conclude that the new dosage
form is effective on the basis of pharmacokinetic data without an
additional clinical efficacy trial.”
The types of studies generally necessary in such cases will depend on the
existence and nature of exposure-response relationships, and whether a
therapeutic window has been established. The following cases provide some
general ideas as to what studies and criteria may need to be met.
There is no prior knowledge of a concentration or exposure—response
relationship or of a therapeutic window; approval is based solely on plasma
profile comparisons and BE comparisons of PK parameters. Generally clinical
trial(s) are necessary for approval in the case where there is no exposure-
response relationship or a therapeutic window. An approach based solely on
pharmacokinetic data with minimum or no information on PK/PD
relationships is not generally encouraged. If it is agreed that the approval
will be entirely based on PK data (e.g., based on prior knowledge of drug or
its extensive use, or another appropriate reason agreed with FDA),
bioequivalence between the IR and ER product is required in terms of Cmax,
Cmin, and AUC at steady state. The overall plasma profile over the ER product’s
dosage interval must also be quite similar to the IR product’s profile over the
same time period. Differences in shapes of the plasma profiles may affect the
efficacy and safety profiles of the drug. In such cases, the differences in shapes
may outweigh findings of BE based on Cmax, Cmin, and AUC. If deviations in
the steady-state PK profiles are seen between the ER and IR product regimens,
additional PK/PD information or clinical studies may be required.
In certain cases, it may also be important to assess differences in steady-
state tmax between the ER and IR products for approval purposes. Additional
BA studies as previously outlined would also be required.
There is no quantitative concentration or exposure-response relationship
but a well-defined therapeutic window in terms of safety and efficacy exists.
1. Case where the rate of input is known not to influence the safety
and efficacy profile: When a therapeutic window that is well

accepted exists and rate of input does not affect the safety/
efficacy profile of the drug, the following criteria may be
appropriate for comparing extended-release products to its
reference (Note: there is no specific FDA Guidance that
addresses this):
• For AUC ss , the 90% confidence interval for the log-
transformed ratio should be between 80–125
• The Cmax ss should be equal to or below the upper limit of the
defined therapeutic window and the absolute Cmin ss should be
equal to or above the lower limit of the defined therapeutic
window.
Additional BA studies as previously outlined would also be
necessary.
2. Case where it is unknown whether the rate of drug input
influences the safety or efficacy profiles of the drug:
Criteria can be the same as subcase 1, but in addition, studies
investigating the impact of the rate of input on the
pharmacodynamics of the drug in terms of safety and efficacy
should be conducted and shown to have no rate effect.
Additional BA studies as previously outlined would also be
necessary.
There is a well-defined quantitative exposure-response relationship shown
using different input rates or developed using the ER product.
1. If a concentration, or exposure-response relationship is
established with the intended clinical endpoint and the safety
profile of the drug is well understood, clinical safety and efficacy
studies on the ER product may not generally be necessary.
Acceptance criteria can be based on predictions of the clinical
response from the steady-state plasma concentration time
profile. Additional BA studies as previously outlined would also
be required.
2. If a concentration, or exposure-response relationship is
established with a validated surrogate measure, which is
accepted as a validated marker for clinical efficacy, and the safety
profile of the drug is well understood, clinical safety and efficacy
studies may not generally be necessary. Acceptance criteria can
be based on predictions of the clinical response from the plasma
concentration profile. Additional BA studies as previously
outlined would also be required.

GENERAL CONSIDERATIONS IN EVALUATING PK/PD RELATIONSHIPS.

In assessing PK/PD relationships, it is important to establish concentration-
effect relationships and to determine the significance of differences in the
shape of the steady-state concentration vs. time profile for an ER product
regimen as compared to the approved IR product regimen. In this regard,
any differential effects based on the rate of absorption and/or the
fluctuation within a profile as related to safety and/or efficacy should be
determined. Issues of tolerance to therapeutic effects and toxic effects
related to drug exposure, concentration, absorption rate, and fluctuation
should also be examined as part of the PK/PD assessment. In certain cases
minimizing fluctuation in a steady-state profile for an ER product may be
desirable to reduce toxicity while maintaining efficacy as compared to the
IR product regimen (e.g., theophylline products). In other cases, minimizing
fluctuation in a steady-state profile for an ER product may reduce efficacy
(e.g., nitroglycerin—due to tolerance) as compared to the IR product
regimen’s profile where higher fluctuation is observed. It is therefore
important to know the profile shape vs. PD relationships.
Safety Assessment of ER Dosage Form

Studies to assess the safety of the ER dosage form are generally necessary.
An example of dosage unit or dosage unit/drug safety problems could be
bezoar formation from some ER formulations.
In vivo Studies Needed for Approval of ER ANDAs

(Generics) [3]
• A single-dose nonreplicate design fasting study comparing the
test and reference-listed drug product. Since single-dose studies
are considered to be most sensitive in addressing the primary
question of bioequivalence [8] i.e., release of the drug at the same
rate and to the same extent, multiple-dose BE studies are no
longer necessary. For extended-release products marketed in
multiple strengths, a single-dose bioequivalence study under
fasting conditions is required only on the highest strength if all
the strengths are proportionally similar and all strengths are
manufactured under the same conditions. Bioequivalence studies
on the lower strengths may be waived based on in vitro
dissolution profiles. If the strengths are not proportionally
similar, a single-dose bioequivalence study is required for each
strength. This requirement can, however, be waived in the
presence of an acceptable in vitro/in vivo correlation [4].

• A fed state nonreplicate design bioequivalence study comparing

the highest strength of the test and reference product [3].
In vitro Studies Needed (Dissolution)

Dissolution testing should be conducted on the ER product batches that
were used in the pivotal BA/BE studies. The dissolution method should be
appropriately selected after evaluation of several dissolution media (different
pH) and agitation speeds, and should have adequate discriminatory power
to differentiate between optimal and suboptimal batches. The sponsors are
encouraged to develop dissolution methods that correlate with in vivo
performance. If bio waivers for lower strengths are requested, adequate
dissolution data needs to be submitted. Details of dissolution testing for ER
products [2–4] can be found in the FDA “Guidance for Industry—Extended
Release Oral Dosage Forms: Development, Evaluation, and Applications of
in vitro/in vivo Correlations.”
POSTAPPROVAL CHANGES
Refer to SUPAC-MR guidance, IVIVC (next section), and biowaivers chapter
for details. In general, when manufacturing changes are made to an approved
extended-release product, e.g., changes in composition, manufacturing site,
batch size, equipment, process, etc., the requirements are defined under the
FDA guidance “Scale-up and post approval changes for modified release
dosage forms” [2]. In cases when the SUPAC-MR Guidance recommends a
biostudy to support the change, an adequate in vitro/in vivo correlation can
be used as justification. These are clearly explained in the FDA guidance on
IVIVC (Extended release oral dosage forms: Development, evaluation and
applications of in vitro/in vivo correlations [4]).
IVIVC [IN VITRO (DISSOLUTION)/IN VIVO (BIOAVAILABILITY)

CORRELATIONS] [4, 9, 12, 13]
Why are IVIVCs Important?

In vitro dissolution has been extensively used as a quality control tool for
solid oral dosage forms. Many times, however, it is not known whether one
can predict the in vivo performance of these products from in vitro dissolution
data. In an effort to minimize unnecessary human testing, investigations of
in vitro/in vivo correlations between in vitro dissolution and in vivo
bioavailability are increasingly becoming an integral part of extended-release
drug product development. This increased activity in developing IVIVCs
indicates the value of IVIVCs to the pharmaceutical industry. Because of the

scientific interest and the associated utility of IVIVC as a valuable tool, the
U.S. Food and Drug Administration has published a Guidance in September
1997, titled Extended Release Oral Dosage Forms: Development, Evaluation
and Applications of in vitro/in vivo Correlations. A predictive IVIVC enables
in vitro dissolution to serve as a surrogate for in vivo bioequivalence testing.
In vitro/in vivo correlations can be used in place of biostudies that may
otherwise be required to demonstrate bioequivalence, when certain changes
are made in formulation, equipment, manufacturing process, or the
manufacturing site. In vitro/in vivo correlation development could lead to
improved product quality (more meaningful dissolution specifications) and
decreased regulatory burden (reduced biostudy requirements).
Principles
In order to successfully develop an IVIVC, dissolution or release from the
formulation has to be the rate-limiting step in the sequence of steps leading
to absorption of the drug into the systemic circulation. Further, to utilize this
dissolution test as a surrogate for bioequivalence (where a relatively simple
in vitro test is used in place of human testing), the IVIVC must be predictive
of in vivo performance of the product.
Levels of Correlation
Four categories of IVIVCs (levels A, B, C, and multiple level C) have been
described in the FDA guidance. In addition, a qualitative rank order
correlation (level D) has also been described in the U.S. Pharmacopoeia.
Level A
A level “A” correlation represents a point-to- point relationship between in
vitro dissolution and the in vivo input rate (e.g., the in vivo dissolution of the
drug from the dosage form). Level A correlation refers to a predictive
mathematical model for the relationship between the entire in vitro
dissolution/release time course and the entire in vivo response time course,
e.g., fraction absorbed vs. fraction dissolved (see Fig. 1). Generally these
correlations are linear; however, nonlinear correlations are also acceptable.
A level “A” correlation is considered to be the most informative and very
useful from a regulatory point of view.
Level B
A level “B” correlation uses the principles of statistical moment analysis
[10]. Level B correlation is a predictive mathematical model of the

FIGURE 1 Level “A” correlation.
relationship between summary parameters (Fig. 2) that characterize the in

vitro and in vivo time courses, e.g.,
a. mean in vitro dissolution time versus mean in vivo dissolution time
b. mean in vitro dissolution time versus mean residence time in vivo
Although this type of correlation uses all of the in vitro and in vivo data, it is
not considered very useful since many different dissolution and plasma
FIGURE 2 Level “B” correlation.

concentration profiles and shapes can give the same mean summary
parameters. Since it does not uniquely reflect the actual in vivo plasma level
curve, this is not very useful from a regulatory point of view.
Level C
A level “C” correlation establishes a single-point relationship between a
dissolution parameter (e.g., time for 50% dissolved or % dissolved in six
hours) and a pharmacokinetic parameter (AUC and Cmax) (Fig. 3).
A level “C” correlation does not reflect the complete shape of the plasma
concentration time curve, therefore is not the most useful correlation from a
regulatory point of view. However, this type of correlation can be useful in
early formulation development.
Multiple Level C
A multiple level “C” correlation relates one or several pharmacokinetic

parameters of interest to the amount of drug dissolved at several time points
of the dissolution profile (e.g., Cmax vs. % dissolved in two hours, six hours,
and 12 hours)—see Fig. 4 below demonstrating a multiple level C
correlation using formulations I to P [11]. This might be accomplished via
linear regression. Multiple level “C” correlation can be as useful as level
“A” IVIVC from a regulatory point of view. However, if one can develop a
multiple level “C” correlation, it is likely that a level “A” correlation can be
developed as well.
FIGURE 3 Level “C” correlation.

FIGURE 4 Multiple level “C” correlation.
When is an IVIVC Likely?

In vitro/in vivo correlations are generally seen when the dissolution or
release of the drug from the dosage form is the rate-limiting step in the
absorption and appearance of the drug in in vivo circulation.
FDA Guidance, “Extended Release Oral Dosage Forms:

Development, Evaluation and Applications of in vitro/in vivo
Correlations” [4]
This guidance has been developed (1) to reduce the regulatory burden by
decreasing the number of biostudies needed to approve and maintain an

extended-release product on the market and (2) to set clinically more

meaningful dissolution specifications. It is anticipated that with a predictive
IVIVC, the biostudies that are generally required for major manufacturing
changes are replaced by a simple in vitro dissolution test.
General Principles/Considerations
The following general considerations apply in the development of an IVIVC:
• Human data are necessary for regulatory consideration of an

IVIVC.
• Bioavailability studies for IVIVC development should be
performed with enough subjects to characterize adequately the
performance of the drug product under study. The number of
subjects in some established IVIVCs has ranged from 6 to 36.
Although crossover studies are preferred, parallel studies or
cross-study analyses (with appropriate normalization with a
common reference) may be acceptable. The reference product in
developing an IVIVC may be an intravenous solution, an
aqueous oral solution, or an immediate-release product.
• In vitro/in vivo correlations should usually be developed in the
fasted state, unless the drug is not tolerated in fasted state and is
indicated to be taken only in fed state due to tolerability
concerns.
• Any in vitro dissolution method may be used to obtain the
dissolution characteristics of the ER dosage form. The most
common dissolution apparatus is USP apparatus I (basket) or II
(paddle), used at compendially recognized rotation speeds (e.g.,
100 rpm for the basket and 50–75 rpm for the paddle). An
aqueous medium, either water or a buffered solution preferably
not exceeding pH 6.8, is recommended as the initial medium for
development of an IVIVC. For poorly soluble drugs, addition of
surfactant (e.g., sodium lauryl sulfate) may be appropriate.
Nonaqueous and hydroalcoholic systems are generally
discouraged. The dissolution profiles of at least 12 individual
dosage units from each lot should be determined.
• Generally, IVIVC should be developed using two or more
formulations with different release rates. When two or more
drug product formulations with different release rates are
developed, their in vitro dissolution profiles should be generated
using an appropriate dissolution methodology. The dissolution
method used should be the same for all the formulations. The
IVIVC relationship should be demonstrated consistently with

two or more formulations with different release rates to result in

corresponding differences in absorption profiles. [9, 12]. When
in vitro dissolution is independent of the dissolution test
conditions (e.g., medium, agitation, pH), development of IVIVC
using one release rate formulation may be sufficient.
• An important factor is the range of release rates to study. The in
vitro and in vivo profiles of the formulations used to develop
IVIVC should be adequately different.
• Dissolution testing can be carried out during the formulation
screening stage using several methods. Once a discriminating
system is developed, dissolution conditions should be the same
for all formulations tested in the biostudy for development of the
correlation and should be fixed before further steps towards
correlation evaluation are undertaken.
• It is important to note that the relationship between in vitro
dissolution and in vivo dissolution, or absorption, should be the
same for all the formulations studied. If one or more of the
formulations (highest or lowest release rate formulations) does
not show the same relationship between in vitro dissolution and
in vivo performance compared with the other formulations, the
correlation may still be used within the range of release rates
encompassed by the remaining formulations.
IVIVC Development
The initial stage of establishing an IVIVC is an exploratory modeling
process. One method to develop a level “A” correlation is to estimate the in
vivo absorption or dissolution time course using an appropriate
deconvolution technique for each formulation and subject (using Wagner-
Nelson method, numerical deconvolution, etc.). The in vivo absorption
profile is plotted against the in vitro dissolution profile to obtain a
correlation (see Figs. 5 and 6).
A Level “A” correlation is usually estimated by a two-stage procedure:
deconvolution followed by comparison of the fraction of drug absorbed to
the fraction of drug dissolved [12]. Details of the deconvolution/
convolution methodology can be found in several literature articles [14–17]
and will not be discussed here. One alternative is based on a convolution
procedure that models the relationship between in vitro dissolution and
plasma concentration in a single step. Plasma concentrations predicted from
the model and those observed are compared directly. For these methods, a
reference treatment is desirable, but the lack of one does not preclude the
ability to develop an IVIVC [16]. Whatever the method used to develop a
Level “A” IVIVC, the IVIVC model should predict the entire in vivo time

FIGURE 5 In vitro dissolution and in vivo profiles.
course from the in vitro data. Here the model refers to the relationship
between in vitro dissolution of an ER dosage form and an in vivo response
such as plasma drug concentration or amount of drug absorbed.
One could use alternative approaches than the ones mentioned to
develop correlations. Also, if there is no one-to-one relationship, then
dissolution conditions may be altered (prior to evaluation of predictability),
or time-scaling approaches [18] may be used to develop the correlation.
However, the time-scaling factor should be the same for all formulations
tested. Different time scales for each of the formulations indicate absence of
an IVIVC.
Evaluation of Predictability of IVIVC (IVIVC Validation)

An IVIVC should be evaluated to demonstrate that the predictability of the
in vivo performance of a drug product, from the in vitro dissolution
characteristics of the drug product formulations, is maintained over a range
of in vitro release rates. A correlation should predict the in vivo performance
accurately and consistently. When such an IVIVC has been established, in
vitro dissolution can be used confidently as a surrogate for in vivo
bioavailability/bioequivalence of ER drug products. Since the focus of
IVIVC evaluation is on the predictive performance of the model, prediction
error is evaluated and used as the criteria for IVIVC evaluation in the FDA
Guidance (Figs. 7 and 8). Depending on the intended application of an
IVIVC and the therapeutic index of the drug, evaluation of predictability
internally and/or externally may be appropriate. Evaluation of internal
predictability is based on the initial data used to develop the IVIVC.
Evaluation of external predictability is based on additional data sets.
External predictability evaluation is not necessary unless the drug is a

Regulatory Considerations
435
FIGURE 6 IVIVC development.
narrow therapeutic index drug, or only two release rates were used to
develop the IVIVC, or if the internal predictability criteria are not met (for
criteria, see p. 438). However, since the IVIVC will potentially be used to
predict the in vivo performance for future changes, it is of value to evaluate
external predictability when additional data are available. An important
concept is that the less data available for initial IVIVC development, the
more additional data may be needed to define completely the IVIVC’s
predictability. Some combination of three or more formulations with
different release rates is considered optimal.
Internal and External Predictability. Estimation of prediction error
internally: Internal predictability should be evaluated for all IVIVCs
(irrespective of the therapeutic index of the drug).
Estimation of prediction error externally. This is appropriate in some
situations, particularly when only two formulations with different release
rates are used to develop the IVIVC model, when calculation of prediction
error internally is inconclusive, or when a narrow therapeutic index drug is
studied.
The additional test data sets used for external prediction error calculation
may have several differing characteristics compared to the data sets used in
IVIVC development. Although formulations with different release rates
provide the optimal test of an IVIVC’s predictability, data from other types
of formulations may be considered. In each case, bioavailability data should
be available for the data set under consideration.
The following represent, in decreasing order of preference, formulations
that may be used to estimate prediction error externally:
• A formulation with a different release rate than those used in

IVIVC development.
• A formulation with the same or similar release rate, but
involving some change in the manufacture of this batch (e.g.,
composition, process, equipment, manufacturing site).
• A formulation with the same or similar release rate obtained
from another batch/lot with no changes in manufacturing.
Methods and Criteria for Evaluation of Predictability. The objective of

IVIVC evaluation is to estimate the magnitude of the error in predicting the
in vivo bioavailability results from in vitro dissolution data. Any
appropriate approach related to this objective may be used for evaluation of
predictability. One approach is to predict the in vivo plasma concentration-
time profile from the in vitro dissolution data. This procedure is shown in
Fig. 7 below, where the in vitro dissolution rate is converted to absorption
rate using the IVIVC model and then convolved to predict the plasma

FIGURE 7 Prediction of in vivo profiles from in vitro dissolution data.
profile. The Cmax and AUC from the predicted profiles should be compared
to those from the observed profile to calculate % prediction errors on Cmax
and AUC (Fig. 8).
Absolute % prediction error on Cmax and AUC:
Internal predictability: The recommended approach involves the use of the

IVIVC model to predict each formulation’s (formulations used in developing

FIGURE 8 Comparsion of observed versus predicted profiles.
the IVIVC) plasma concentration profile (or Cmax and/or AUC for a multiple
level C IVIVC) from each respective formulation’s dissolution data.
Calculate the % prediction error on Cmax and AUC.
Criteria
• Average absolute percent prediction error (% PE) of 10% or less

for Cmax and AUC establishes the predictability of the IVIVC. In
addition, the % PE for each formulation should not exceed 15%.
• If these criteria are not met, that is, if the internal predictability
of the IVIVC is inconclusive, evaluation of external
predictability of the IVIVC should be performed as a final
determination of the ability of the IVIVC to allow the use of in
vitro dissolution as a surrogate for bioequivalence.
External predictability: This involves using the IVIVC to predict the in vivo
performance of a formulation with known bioavailability that was not used
in developing the IVIVC model.
Criteria
• The percent prediction error of 10% or less for Cmax and AUC
establishes the external predictability of an IVIVC.
• The percent prediction error between 10 and 20% indicates
inconclusive predictability and the need for further study using
additional data sets. Results of estimation of PE from all such
data sets should be evaluated for consistency of predictability.
• The percent prediction error greater than 20% generally
indicates inadequate predictability

Caution During Evaluation of Predictability

In the evaluation of internal predictability, it is recommended that the PK
parameter estimates used (e.g., for unit impulse response) in predicting the
in vivo performance should be the average values or population estimates.
Individual PK parameters should not be used to predict individual PK
profiles which then are averaged to obtain the predicted average
concentration—time profiles. This is due to the following three problems:
1. One does not have dissolution data on the dosage unit that the
individual subject was administered. Therefore the input
function is based on average parameters. Use of average in vitro
parameters and individual in vivo parameters is not appropriate.
2. The percent prediction error calculated in this manner for
internal predictability will always look better since the IVIVC
was developed using the same individual values, and one is
trying to predict the same data using the same individual
estimates.
3. Further, since IVIVC will be used to obtain bio waivers when
changes are made in future, based on in vitro dissolution data
(and no in vivo data), one does not know what the individual
parameters will be in each patient that is likely to use the drug.
Therefore use of population estimates or mean PK parameters is
recommended.
Applications of IVIVC
A predictive IVIVC can empower in vitro dissolution to act as a surrogate
for in vivo bioavailability/bioequivalence. This can be used to grant
biowaivers and to set meaningful dissolution specifications that take into
account the clinical consequences.
Biowaivers. The Guidance outlines five categories of biowaivers. These
are described in detail below.
1. Biowaivers without an IVIVC.

2. Biowaivers using an IVIVC: Nonnarrow therapeutic index
drugs.
3. Biowaivers using an IVIVC: Narrow therapeutic index drugs.
4. Biowaivers when in vitro dissolution is independent of
dissolution test conditions.
5. Situations for which an IVIVC is not recommended for
biowaivers.

Ideally, one would like to be able to predict the in vivo performance of the
drug product from its in vitro dissolution. Therefore, with a predictive
IVIVC, waivers for in vivo bioavailability studies may be granted for
manufacturing site changes, equipment changes, manufacturing process
changes, and formulation composition changes. The biowaivers section
deals with changes ranging from situations such as minor changes, which
are insignificant for product performance, to major changes for which an
IVIVC is not sufficient to justify the change, for a regulatory decision. The
IVIVC guidance in this area complements the SUPAC-MR guidance (Scale
Up and Post Approval Changes—Modified Release Dosage Forms) [2]. An
IVIVC can be used to support those drug product changes in SUPAC-MR
that might have required a biostudy. However, there are situations such as
those outlined under category 5, where an IVIVC cannot be used.
The mechanism of drug release from the drug product should remain the
same when changes are made to a formulation for an IVIVC to be
applicable. If the release mechanism changes (e.g., from a diffusion-
controlled release to an osmotic release; beads to a matrix tablet), a
previously developed IVIVC is not applicable.
The two criteria for granting a biowaiver for a new formulation, where
an IVIVC has been established, are that the differences in predicted means of
Cmax and AUC are no more than 20% from that of the reference product
and, where applicable, the new formulation meets the application or
compendial dissolution specifications (see Fig. 9).
FIGURE 9 Prediction of in vivo profiles using IVIVC to grant biowaivers.

Biowaivers with and without an IVIVC

Category 1: Biowaivers Without an IVIVC
This section relates to waivers for lower strengths (beaded
capsules as well as tablets), changes made to lower strengths and
certain preapproval changes—see biowaivers chapter and IVIVC
Guidance for details.
Category 2: Biowaivers Using an IVIVC: Nonnarrow Therapeutic

Index Drugs [4]
a. Two Formulations/Release Rates

A biowaiver is possible for an ER drug product using an IVIVC
developed with two formulations/release rates for (1) Level 3
manufacturing site changes as defined in SUPAC-MR and (2)
Level 3 nonrelease controlling excipient changes as defined in
SUPAC-MR, with the exception of complete removal or
replacement of excipients (see below).
b. Three Formulations/Release Rates
developed with three formulations/release rates (or developed with
two formulations/release rates with establishment of external
predictability) for (1) Level 3 process changes as defined in SUPAC-
MR; (2) complete removal of or replacement of nonrelease controlling
excipients as defined in SUPAC-MR; and (3) Level 3 changes in the
release controlling excipients as defined in SUPAC-MR.
c. Biowaivers for Lower Strengths
If an IVIVC is developed with the highest strength, waivers for
changes made on the highest strength and any lower strengths
may be granted if these strengths are compositionally proportional
or qualitatively the same, the in vitro dissolution profiles of all
the strengths are similar, and all strengths have the same release
mechanism.
d. Biowaiver for New Strengths
This biowaiver is applicable generally to strengths lower than the
highest strength (in some instances under an NDA (such as for
compositionally proportional formulations), waiver for higher
strengths may be possible if scientifically justified especially
using an established IVIVC). For details on biowaiver and
criteria for new strengths (in an NDA or an ANDA as a generic),
see biowaivers chapter.
e. Changes in Release-Controlling Excipients
Changes in release-controlling excipients in the formulation should
be within the quantitative range of release-controlling excipients

(used in the different release rate formulations) of the established

correlation.
f. Obtaining Category 2a, 2b, and 2c Biowaivers: The difference in
predicted means of Cmax and AUC should be no more than 20%
from that of the reference product and, where appropriate, the
new formulation should meet the application/compendial
dissolution specifications.
Category 3: Biowaivers Using an IVIVC: Narrow Therapeutic

Index Drugs [4]
If external predictability of an IVIVC is established, the following
waivers (all waivers described under category 2 above including
major site changes and nonrelease-controlling excipient changes)
are possible if at least two formulations/release rates have been
studied for the development of the IVIVC.
a. Manufacturing changes
for (1) Level 3 process changes as defined in SUP AC-MR; (2)
complete removal of or replacement of nonrelease-controlling
excipients as defined in SUP AC-MR; and (3) Level 3 changes in
the release-controlling excipients as defined in SUPAC-MR.
b. Biowaivers for Lower Strengths—see category 2c above for
details
c. Approval of New Strengths—see category 2d above for details
Obtaining category 3c biowaivers: see requirements for
obtaining 2d biowaivers
d. Changes in Release-Controlling Excipients—see category 2e
above
e. Obtaining Category 3a and 3b Biowaivers: see requirements
under category 2f above.
Category 4: Biowaivers When In Vitro Dissolution Is Independent

of Dissolution Test Conditions [4]
Situations in which biowaivers are likely to be granted for both
narrow and nonnarrow therapeutic index drugs:
a. Categories 2 and 3 biowaivers are likely to be granted with an

IVIVC established with one formulation/release rate.
b. Obtaining Category 4 Biowaivers
• Biowaivers may be granted if dissolution data are submitted
in application/compendial medium and in three other media
(e.g., water, 0.1 NHCl, USP buffer at pH 6.8) and the in vitro
dissolution is shown to be independent of dissolution test

conditions after the change is made in drug product

manufacturing.
• The difference in predicted means of Cmax and AUC should
be no more than 20% from that of the reference product
and, where appropriate, the new formulation should meet
the application/compendial dissolution specifications. For
new strengths, see 2d above.
Category 5: Situations for which an IVIVC Is Not Recommended [4]
a. Approval of a new formulation of an approved ER drug product

when the new formulation has a different release mechanism.
b. Approval of a dosage strength higher or lower than the doses
that have been shown to be safe and effective in clinical trials.
c. Approval of another sponsor’s ER product even with the same
release-controlling mechanism.
d. Approval of a formulation change involving a nonrelease-
controlling excipient in the drug product that may significantly
affect drug absorption.
Setting Dissolution Specifications [4]. Once an IVIVC is developed, this

should be used to set dissolution specifications for the product. An IVIVC
provides in vivo relevance to in vitro dissolution specifications, beyond
batch-to-batch quality control. In this approach, the in vitro dissolution test
becomes a meaningful predictor of in vivo performance of the formulation,
and dissolution specifications may be used to minimize the possibility of
releasing lots that would be different in in vivo performance.
1. Setting Dissolution Specifications Without an IVIVC
• The recommended range for dissolution specifications at any

time point is ±10% of the label claim deviation from the mean
dissolution profile obtained from the clinical/bioavailability
batches. In certain cases, reasonable deviations from the ±10%
range can be accepted provided that the range at any time point
does not exceed 25%. Specifications greater than 25% may be
acceptable based on evidence that lots (side batches) with mean
dissolution profiles that are allowed by the upper and lower
limits of the specifications are bioequivalent.
• A minimum of three time points are recommended to set the
specifications. These time points should cover the early, middle,
and late stages of the dissolution profile. The last time point

should be the time point where at least 80% of drug has

dissolved, or the time when the plateau of the dissolution profile
has been reached.
• Specifications should be established based on average dissolution
data (n= 12) for each lot under study, equivalent to USP Stage 2
testing. Specifications that allow all lots to pass at Stage 1 of
testing may result in lots with less than optimal in vivo
performance passing these specifications at USP Stage 2 or Stage 3.
USP acceptance criteria for dissolution testing are recommended
unless alternate acceptance criteria are specified in the ANDA/
NDA.
2. Setting Dissolution Specifications Where an IVIVC Has Been Established

If an IVIVC has been established, it should be used to set dissolution
specifications. Optimally, specifications should be established such that all
lots that have dissolution profiles within the upper and lower limits of the
specifications are bioequivalent. Less optimally but still possible, lots
exhibiting dissolution profiles at the upper and lower dissolution limits
should be bioequivalent to the clinical/bioavailability lots or to an
appropriate reference standard.
a. Level A Correlation Established
• Specifications should be established based on average data (n=12).
• A minimum of three time points that cover the early, middle, and
late stages of the dissolution profile is recommended to establish
the specifications. The last time point should be the time point
where at least 80% of drug has dissolved or the time where the
plateau of the dissolution profile has been reached.
• Predict the plasma concentration time profile using convolution
techniques or other appropriate modeling techniques and
determine whether the lots with the fastest and slowest release
rates that are allowed by the dissolution specifications result in a
maximal difference of 20% in the predicted Cmax and AUC (see
Fig. 10). An established IVIVC may allow setting wider
dissolution specifications. This would be dependent on the
predictions of the IVIVC (i.e., 20% differences in the predicted
Cmax and AUC). However, if based on the IVIVC, the dissolution
specifications justified are less than the 20% range allowed with
an IVIVC, a minimum range of 20% will be generally allowed
unless there are clinical concerns.
• USP acceptance criteria for dissolution testing are recommended
unless alternate acceptance criteria are specified in the ANDA/
NDA.

FIGURE 10 Setting dissolution specifications based on level “A” IVIVC.
b. Multiple Level C Correlation Established

If a multiple-point Level C IVIVC has been established, establish the
specifications at each time point such that there is a maximal difference of
20% in the predicted Cmax and AUC. Additionally, the last time point should
be the time point where at least 80% of drug has dissolved.
c. Level C Correlation Based on Single Time Point Established

This one time point may be used to establish the specification such that there
is not more than a 20% difference in the predicted AUC and Cmax. At other
time points, the maximum recommended range at any dissolution time
point specification should be ±10% of label claim deviation from the mean
dissolution profile obtained from the clinical/bioavailability lots.
Reasonable deviations from ± 10% may be acceptable if the range at any
time point does not exceed 25%.
3. Setting Specifications Based on Release Rate

If the release characteristics of the formulation can be described by a zero-
order process for some period of time (e.g., 5%/hr from 4 to 12 hours), and
the dissolution profile appears to fit a linear function for that period of time,
a release-rate specification may be established to describe the dissolution
characteristics of that formulation. Such a specification may provide for a
better control of the in vivo performance of the product. A release rate
specification may be (i) an addition to the specifications established on the
cumulative amount dissolved at the selected time points, or (ii) may be the
only specification along with a cumulative dissolution specification for time
when at least 80% of drug has dissolved.

Regulatory Impact of IVIVCs

IVIVC can impart in vivo meaning to the in vitro dissolution test and can be
useful as surrogate for bioequivalence. IVIVCs can thus decrease regulatory
burden by decreasing the number of biostudies required in support of a drug
product. As an additional benefit to the sponsors, IVIVC can support wider
in vitro dissolution specifications, where justified. FDA strongly encourages
the development and evaluation of IVIVCs during ER product development.
Generally IVIVC development adds value to the overall drug development
process by providing an understanding of the relevance of the in vitro
dissolution data leading to better utilization of the in vitro dissolution test.
Usually this IVIVC development can be done without conducting new
studies. One can use the early development studies where multiple release-
rate formulations are generally incorporated in the bioavailability studies.
IVIVCs can thus be useful in decreasing the regulatory burden with no
undue penalty to the companies that develop these correlations.
EMEA GUIDANCE THAT DEALS WITH IVIVC [19]

The EMEA Guidance on Quality of MR products and transdermal products
covers some of the considerations in development and evaluation of IVIVC
and some applications of IVIVC. Similar to U.S. FDA, sponsors are asked to
consider development of an IVIVC. If an IVIVC is established, the
dissolution test, after proper validation, can be used as a “qualifying control
method with in vivo relevance” rather than just a quality control test.
• Levels of correlations are defined in a similar manner to the FDA

Guidance.
• Development of IVIVC: Development considerations of levels A,
B, and C IVIVC are briefly discussed in this guidance. For a level
A IVIVC, generally one formulation tested at different
dissolution conditions should be compared to aqueous solution.
This seems to be different (although not explicit) from the FDA
Guidance where there is a need to study multiple release-rate
formulations.
• Evaluation of predictability: Methods and criteria for
predictability are the same as in the FDA Guidance; however,
there is no explicit discussion of situations with condition-
independent dissolution or narrow therapeutic index drugs.
• Applications—Biowaivers: While the FDA Guidance provides
detailed situations for biowaivers, the EMEA Guidance provides
a summary to state that when a Level A IVIVC has been
established and the release specification is not changed, type II

variations (e.g., major changes in nonrelease-controlling

excipients, insignificant changes in release-controlling excipients
or major changes in method of manufacturing) may be accepted
on the basis of in vitro data, the therapeutic index of the drug
substance and predictability of the IVIVC. In general, BA/BE
data are needed for products with an established level B or C
correlation or no IVIVC, unless justified.
• Applications—Dissolution specifications: If IVIVC is established,
it is used to set specifications. However, there are some differences
from the FDA Guidance.
(A) Level A: The specification is based on a 1:1 correlation between
the dissolution profile in vivo and in vitro (FDA Guidance is not
restricted to a 1:1 correlation).
(B) Level B correlation can also be used to set specifications,
although methodology details are not provided (Level B
correlations are not useful for waivers or setting dissolution
specifications according to the FDA Guidance).
(C) For any level of correlation, i.e., levels A, B, C, or multiple
level C, specifications should be set such that the maximal
difference in predicted AUC is 20% and, predicted Cmax only if
relevant (FDA Guidance requires both AUC and Cmax).
REFERENCES
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Controlled Release Products, Controlled Release Drug Delivery Systems:
Scientific and Regulatory Issues, Fifth International Symposium on Drug
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Forms: Scale-Up and Post-Approval Changes: Chemistry, Manufacturing and
Controls, in vitro Dissolution Testing, and in vivo Bioequivalence
Documentation, September 1997.
3. FDA, Guidance for Industry: Bioavailability and Bioequivalence Studies for
Orally Administered Drug Products—General Considerations, March 2003.
4. FDA, Guidance for Industry: Extended Release Oral Dosage Forms:
Development, Evaluation, and Application of in vitro/in vivo Correlations,
September 1997.
5. Code of Federal Regulations 21 section 320.
6. FDA, Guidance for Industry: Food-effect Bioavailability and Fed Bioequivalence
Studies, December 2002.
7. FDA, Guidance for Industry: Providing Clinical Evidence of Effectiveness for
Human Drug and Biological Products, May 1998.

8. El-Tahtawy, A.A.; Jackson, A.J.; Ludden, T.M. Comparison of Single and

Multiple Dose Pharmacokinetics Using Clinical Bioequivalence Data and Monte
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10. Yamaoka, K.; Nakagawa, T.; Uno, T. Statistical Moments in Pharmacokinetics.
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Evaluation and Research, Food and Drug Administration, June 1991.
12. Eddington, N.D.; Marroum, P.; Uppoor, R.; Hussain, A.; Augsburger, L.
Development and Internal Validation of an in vitro-in vivo Correlation for
Hydrophilic Metoprolol Tartrate Extended Release Tablet Formulations.
Pharmaceutical Research 1998, 15, 464–471.
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Augsburger, L.L.; Eddington, N.D. Evaluation of External Predictability of an in
vitro-in vivo Correlation for an Extended-Release Formulation Containing
Metoprolol Tartrate. Journal of Pharmaceutical Sciences, 2000, 89(10), 1354–
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(11) 1604–1607.
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Forms Section 1 (Quality), 29 July 1999.

19
In vivo Bioavailability/Bioequivalence
Waivers
Patrick J.Marroum, Ramana S.Uppoor,

and Mehul U.Mehta
INTRODUCTION
Bioavailability (BA) is defined in 21 CFR 320.1 as “the rate and extent to

which the active ingredient or active moiety is absorbed from a drug product
and becomes available at the site of action. For drug products that are not
intended to be absorbed into the bloodstream [1], bioavailability may be
assessed by measurements intended to reflect the rate and extent to which
the active ingredient or active moiety becomes available at the site of action.”
Bioequivalence (BE) is defined in 21 CFR 320.1 as “the absence of a
significant difference in the rate and extent to which the active ingredient
or active moiety in pharmaceutical equivalents or pharmaceutical
alternatives becomes available at the site of drug action when administered
at the same molar dose under similar conditions in an appropriately designed
study.” As noted in the statutory definitions, both BE and product quality
BA focus on the release of a drug substance from a drug product and
subsequent absorption into the systemic circulation [1]. Over the last 30
449
450 Marroum et al.
years, dissolution testing has not only been recognized as a valuable quality
control test but has also proved itself as a useful indicator of differences in
bioavailability. This is due to the fact that drug absorption after oral
administration depends on the release of the drug substance from the drug
product, the dissolution or solubilization of the drug under physiological
conditions and the permeability across the gastrointestinal tract. Whenever,
a significant difference in bioavailability has been found among supposedly
identical articles, the dissolution test most of the times has been able to
discriminate among these articles. In fact, dissolution is so sensitive to
formulation factors that bioequivalent formulations sometimes show
differences in dissolution profiles. According to the regulations stated in
CFR 320.24, bioavailability and bioequivalence could be assessed by several
in vitro or in vivo methods depending on the purpose of the study, the
availability of analytical methods, and the nature of the drug product.
Specifically CFR 320.24 states that either an in vitro test that has been
correlated with and is predictive of human bioavailability data or a currently
available in vitro test acceptable to FDA that ensures that human in vivo
bioavailability is acceptable [2]. This chapter starts with definitions followed
by the relevant regulations governing in vivo bioavailability/bioequivalence
waivers with a discussion on the various types of waivers based on
comparability of dissolution profiles for both immediate-release (IR) dosage
forms and modified-release (MR) dosage forms. Moreover, the types of
scale up and postapproval changes that can be approved based on
comparability of dissolution profiles are summarized for both IR and MR
products. A brief description on how to compare dissolution profiles is
given. The role of in vitro-in vivo correlations (IVIVC) for MR products as
well as the biopharmaceutics classification system (BCS) for IR products in
alleviating the regulatory burden is elucidated. Finally, an overview of the
Japanese, European, and Canadian guidelines for instances where an in
vivo BA/BE waiver can be granted based on comparability of dissolution
profiles is provided.
DEFINITIONS
Proportionally Similar.
Definition 1: All active and inactive ingredients are in exactly the same
proportion between different strengths (e.g., a tablet of 50-mg strength has
all the inactive ingredients, exactly half that of a tablet of 100-mg strength,
and twice that of a tablet of 25-mg strength).
Definition 2: Active and inactive ingredients are not in exactly the same
proportion between different strengths as stated above, but the ratios of

In vivo Bioavailability/Bioequivalence Waivers 451
inactive ingredients to total weight of the dosage form are within the limits
defined by the SUPAC-IR and SUP AC-MR guidances up to and including
Level II.
Definition 3: For high potency drug substances, where the amount of the
active drug substance in the dosage form is relatively low, the total weight of
the dosage form remains nearly the same for all strengths (within ±10% of
the total weight of the strength on which a biostudy was performed), the
same inactive ingredients are used for all strengths, and the change in any
strength is obtained by altering the amount of the active ingredients and one
or more of the inactive ingredients. The changes in the inactive ingredients
are within the limits defined by the SUPAC-IR and SUPAC-MR guidances
up to and including Level II [3].
Delayed Release: As defined in the U.S. Pharmacopeia (USP), delayed-
release drug products are dosage forms that release the drugs at a time later
than immediately after administration (i.e., these drug products exhibit a lag
time in quantifiable plasma concentrations) [4].
Extended-Release: These are dosage forms that allow a reduction in dosing
frequency as compared to when the drug is present in an immediate-release
dosage form. These drug products can also be developed to reduce fluctuations
in plasma concentrations. Extended-release products can be capsules, tablets,
granules, pellets, and suspensions [4].
Case A Dissolution: Amount dissolved equals 85% in 15 minutes in 900
mL of 0.1 N HC1 using USP apparatus 1 at 100 rpm or apparatus 2 at 50
rpm.
Case B Dissolution: Multipoint dissolution profile in the application/
compendial medium at 15, 30, 45, 60, and 120 minutes or until either 90%
of the drug from the drug product is dissolved or an asymptote is reached
for the proposed and currently accepted formulation.
Case C Dissolution: Multipoint dissolution profiles performed in water,
0.1N HC1, and USP buffer at pH 4.5, 6.5, and 7.5 (five separate profiles)
for the proposed and currently accepted formulations. Adequate sampling
should be performed at 15, 30, 45, 60, and 120 minutes until either 90% of
the drug from the drug product is dissolved or an asymptote is reached. A
surfactant may be used with appropriate justification [5].
Pharmaceutical Equivalents: Drug products are considered pharmaceutical
equivalents if they contain the same active ingredient(s), are of the same
dosage form and route of administration, and are identical in strength and
concentration [6].
Therapeutic Equivalents: Drug products are considered to be therapeutic
equivalents only if they are pharmaceutical equivalents and if they can be
expected to have the same clinical effect and safety profile when administered
to patients under the conditions specified in the label.
Pharmaceutical Alternatives: Drug products are considered pharmaceutical

452 Marroum et al.
alternatives if they contain the same therapeutic moiety or are different dosage
forms or strengths [6].
CODE OF FEDERAL REGULATIONS
CFR 320.22 [7] gives FDA the authority under certain circumstances to waive
the requirements for evidence for determining the in vivo bioavailability and
bioequivalence. Specifically the CFR states:
a. Any person submitting a full or abbreviated new drug application,

or a supplemental application proposing any of the changes set
forth in Sec. 320.21(c), may request FDA to waive the requirement
for the submission of evidence demonstrating the in vivo
bioavailability or bioequivalence of the drug product that is the
subject of the application. An applicant shall submit a request for
waiver with the application. Except for certain situations, FDA
shall waive the requirement for the submission of evidence of in
vivo bioavailability or bioequivalence if the drug product meets
any of the provisions of paragraphs (b), (c), (d), or (e) of this
section.
b. For certain drug products, the in vivo bioavailability or
bioequivalence of the drug product may be self-evident. FDA shall
waive the requirement for the submission of evidence obtained in
vivo demonstrating the bioavailability or bioequivalence of these
drug products. A drug product’s in vivo bioavailability or
bioequivalence may be considered self-evident based on other data
in the application if the product meets one of the following criteria:
1. The drug product:
i. Is a parenteral solution intended solely for administration
by injection, or an ophthalmic or otic solution; and
ii. Contains the same active and inactive ingredients in the
same concentration as a drug product that is the subject
of an approved full new drug application.
i. Is administered by inhalation as a gas, e.g., a medicinal
or an inhalation anesthetic; and
ii. Contains an active ingredient in the same dosage form
as a drug product that is the subject of an approved full
new drug application.


i. Is a solution for application to the skin, an oral solution,
elixir, syrup, tincture, or similar other solubilized form.
ii. Contains an active drug ingredient in the same
concentration and dosage form as a drug product that is
the subject of an approved full new drug application;
and
iii. Contains no inactive ingredient or other change in
formulation from the drug product that is the subject of
the approved full new drug application that may
significantly affect absorption of the active drug
ingredient or active moiety.
c. FDA shall waive the requirement for the submission of evidence
demonstrating the in vivo bioavailability of a solid oral dosage
form (other than an enteric coated or controlled-release dosage
form) of a drug product determined to be effective for at least
one indication in a Drug Efficacy Study Implementation notice
or which is identical, related, or similar to such a drug product
under Sec. 310.6 of this chapter unless FDA has evaluated the
drug product under the criteria set forth in Sec. 320.32, included
the drug product in the Approved Drug Products with Therapeutic
Equivalence Evaluations List, and rated the drug product as having
a known or potential bioequivalence problem. A drug product so
rated reflects a determination by FDA that an in vivo bioequi
valence study is required.
d. For certain drug products, bioavailability or bioequivalence may
be demonstrated by evidence obtained in vitro in lieu of in vivo
data. FDA shall waive the requirement for the submission of
evidence obtained in vivo demonstrating the bioavailability of
the drug product if the drug product meets one of the following
criteria:
1. The drug product is in the same dosage form, but in a different
strength, and is proportionally similar in its active and inactive
ingredients to another drug product for which the same
manufacturer has obtained approval and the conditions in
paragraphs (d)(2)(i) through (d)(2)(iii) of this section are met:
i. The bioavailability of this other drug product has been
demonstrated,
ii. Both the drug products meet an appropriate in vitro test
approved by FDA.
iii. The applicant submits evidence showing that both drug

454 Marroum et al.
products are proportionally similar in their active and

inactive ingredients.
iv. This subparagraph does not apply to enteric coated or
controlled-release dosage forms.
2. The drug product is, on the basis of scientific evidence
submitted in the application, shown to meet an in vitro test
that has been correlated with in vivo data.
3. The drug product is a reformulated product that is identical,
except for a different color, flavor, or preservative that could
not affect the bioavailability of the reformulated product, to
another drug product for which the same manufacturer has
obtained approval and the following conditions are met:
i. The bioavailability of the other product has been
demonstrated,
ii. Both drug products meet an appropriate in vitro test
approved by FDA.
e. FDA, for good cause, may waive a requirement for the submission
of evidence of in vivo bioavailability if waiver is compatible with
the protection of the public health. For full new drug applications,
FDA may defer a requirement for the submission of evidence of
in vivo bioavailability if deferral is compatible with the protection
of the public health.
f. FDA, for good cause, may require evidence of in vivo
bioavailability or bioequivalence for any drug product if the
agency determines that any difference between the drug product
and a listed drug may affect the bioavailability or bioequivalence
of the drug product.
WAIVERS OF IN VIVO BIOAVAILABILITY/BIOEQUIVALENCE

STUDIES WITHOUT IVIVC
Different Strengths
Immediate-release Drug Products
When the drug product is in the same dosage form, but in a different strength,
and is proportionally similar in its active and inactive ingredients to that of
a listed drug, an in vivo BE demonstration of one or more lower strengths
can be waived based on dissolution tests and an in vivo study on the highest
strength.

For an NDA, biowaivers of a higher strength will be determined to be

appropriate based on (1) clinical safety and/or efficacy studies including data
on the dose and the desirability of the higher strength; (2) linear elimination
kinetics over the therapeutic dose range; (3) the higher strength being
proportionally similar to the lower strength; and (4) the same dissolution
procedures being used for both strengths, and similar dissolution results
obtained in the approved medium. If the dissolution medium has not been
selected, then dissolution profiles in three media should be generated (0.1 N
HC1, phosphate buffer pH 4.5 and 6.8). A dissolution profile should be
generated for all strengths [3].
For an ANDA, conducting an in vivo study on a strength that is not the
highest may be appropriate for reasons of safety, subject to approval by
review staff. In addition, as with an NDA, the Agency will consider a waiver
request for a recently approved higher strength when an in vivo BE study
was performed on a lower strength of the same drug product submitted in
an ANDA under the following circumstances:
• Linear elimination kinetics has been shown over the therapeutic

dose range.
• The higher strength is proportionally similar to the lower strength.
• Comparative dissolution testing on the higher strength of the
test and reference drug product is submitted and found
acceptable.
• The sponsor initiated the BE study on the lower strength within
five working days of the approval date of a higher strength of the
reference-listed drug. A study is considered initiated when the
first subject is dosed.
Sponsors of AND As wishing to submit a biowaiver request under these

circumstances should first contact the Regulatory Support Branch, Office of
Generic Drugs, for advice on the proper filing procedure.
Modified-release Drug Products

Beaded Capsules—Lower Strength. For extended-release beaded capsules,
where the strength differs only in the number of beads containing the active
moiety, a single-dose, fasting BE study can be carried out only on the highest
strength, with a request for a waiver of in vivo studies for lower strengths
based on dissolution profiles. A dissolution profile should be generated for
each strength using the recommended dissolution method. The f2 test should
be used to compare profiles from the different strengths of the product. An f2
value of ⱖ 50 can be used to confirm that further in vivo studies are not
needed.

456 Marroum et al.
Tablets—Lower Strength. For extended-release tablets, when the drug

product is in the same dosage form but in a different strength, is proportionally
similar in its active and inactive ingredients, and has the same drug-release
mechanism, an in vivo BE determination of one or more lower strengths can
be waived based on dissolution profile comparisons, with an in vivo study
only on the highest strength. The drug products should exhibit similar
dissolution profiles between the highest strength and the lower strengths
based on the f2 test in at least three dissolution media (e.g., pH 1.2, 4.5, and
6.8). The dissolution profile should be generated on the test and reference
products of all strengths [3].
Transdermal Patches
In vivo bioavailability/bioequivalence demonstration for lower strengths
transdermal patches can be waived based on comparability of dissolution
profiles in three media (0.1 N HC1, phosphate buffer pH 4.5 and 6.8) and
the presence of an acceptable in vivo study on the highest strengths, provided
that the lower strengths patches are compositionally proportional in all their
components and are manufactured under the same manufacturing conditions
at the same manufacturing site using the same equipment as in the case of
highest strengths.
Clinical vs. Market Formulation

During the course of drug development, sponsors sometimes have to blind
the formulations that they use in the clinical trials. In certain situations, the
only difference between the market and clinical trial formulation is that the
tablet mix or the tablet itself is put into a capsule. This is done mainly for
blinding purposes. It is thus possible to get a waiver for the bioequivalence
study that links the market and clinical trial formulation, provided that no
other excipients are added to the capsule that are known to affect the release
of the active drug from the capsule. The waiver of this in vivo bioequivalence
study is granted based on the comparability of the dissolution profile in
three media: 0.1 N HC1 and phosphate buffer pH 4.5 and 6.8.
Scale Up and Postapproval Changes

It is possible that postapproval and sometimes preapproval, a sponsor might
make certain formulation changes in components and composition, scale up
change, manufacturing site change, and manufacturing process or equipment
change. Depending on the possible impact of the manufacturing change on
the release of the active ingredient and its bioavailability from that
formulation, certain manufacturing changes can be approved solely based

on comparability of the dissolution profiles between the changed and

unchanged formulation. Both guidances on Scale Up and Postapproval
Changes for immediate-release formulations and for modified-release
formulations define three levels of change.
According to these guidances, a level 1 change is a change that is unlikely
to have any detectable impact on formulation quality and performance [5].
A level 2 change is defined as a change that could have a significant impact
on formulation quality and performance. The amount of information required
for the approval of such changes depends on the therapeutic window of the
drug, its solubility, and permeability.
Level 3 changes are defined as changes that are likely to have a significant
impact on formulation quality and performance.
In general, level 1 and 2 changes can be approved based on comparability
of dissolution profiles while level 3 changes usually necessitate an in vivo
bioequivalence study.
Tables 1 and 2 summarize the type of change that can be approved
just based on in vitro dissolution data for IR and MR formulations,
respectively [8].
TABLE 1 Summary of the in vitro Dissolution Data Requirements for the

Manufacturing Changes for Immediate-Release Formulations for which an in vivo
Bioavailability Waiver can be Obtained

458 Marroum et al.
TABLE 2 Summary of the In vitro Dissolution Data Requirements for the

Manufacturing Changes for Modified-Release Formulations for which an in vivo
Bioavailability Waiver can be Obtained
DISSOLUTION PROFILE COMPARISONS
Dissolution profiles may be considered similar by virtue of (1) overall profile

similarity and (2) similarity at every dissolution sample time point. The
dissolution profile comparison may be carried out using model-independent
or model-dependent methods.

Model-independent Approach Using a Similarity Factor

A simple model-independent approach uses a difference factor (f1) and a
similarity factor (f2) to compare dissolution profiles [5]. The difference factor
(f1) calculates the percent (%) difference between the two curves at each time
point and is a measurement of the relative error between the two curves:
where n is the number of time points, Rt is the dissolution value of the reference
(prechange) batch at time t, and Tt is the dissolution value of the test
(postchange) batch at time t.
The similarity factor (f 2) is a logarithmic reciprocal square root
transformation of the sum of squared error and is a measurement of the
similarity in the percent (%) dissolution between the two curves.
A specific procedure to determine difference and similarity factors is as

follows:
1. Determine the dissolution profile of two products (12 units each)
of the test (postchange) and reference (prechange) products.
2. Using the mean dissolution values from both the curves at each
time interval, calculate the difference factor (f1) and similarity
factor (f2) using the above equations.
3. For curves to be considered similar, f1 values should be close to 0,
and f2 values should be close to 100. Generally, f1 values up to 15
(0–15) and f2 values greater than 50 (50–100) ensure sameness
or equivalence of the two curves and, thus, of the performance of
the test (postchange) and reference (prechange) products.
This model-independent method is most suitable for dissolution profile
comparison when three to four or more dissolution time points are available.
As further suggestions for the general approach, the following
recommendations should also be considered: The dissolution measurements
of the test and reference batches should be made under exactly the same
conditions. The dissolution time points for both the profiles should be the
same (e.g., 15, 30, 45, and 60 minutes). The reference batch used should be
the most recently manufactured prechange product. Only one measurement

460 Marroum et al.
should be considered after 85% dissolution of both the products. To allow

use of mean data, the percent coefficient of variation at the earlier time points
(e.g., 15 minutes) should not be more than 20%, and at other time points
should not be more than 10%. The mean dissolution values for R can be
derived either from (1) last prechange (reference) batch or (2) last two or
more consecutively manufactured prechange batches.
Model-Independent Multivariate Confidence Region Procedure

In instances where within batch variation is more than 15% CV, a multivariate
model-independent procedure is more suitable for dissolution profile
comparison. The following steps are suggested:
1. Determine the similarity limits in terms of multivariate statistical

distance (MSD) based on interbatch differences in dissolution from
reference (standard approved) batches.
2. Estimate the MSD between the test and reference mean
dissolutions.
3. Estimate 90% confidence interval of true MSD between test and
reference batches.
4. Compare the upper limit of the confidence interval with the
similarity limit.
The test batch is considered similar to the reference batch if the upper limit
of the confidence interval is less than or equal to the similarity limit.
Model-Dependent Approaches
Several mathematical models have been described in the literature to fit
dissolution profiles. To allow application of these models to comparison of
dissolution profiles, the following procedures are suggested:
1. Select the most appropriate model for the dissolution profiles from
the standard, prechange, approved batches. A model with no more
than three parameters (such as linear, quadratic, logistic, probit,
and Weibull models) is recommended.
2. Using data for the profile generated for each unit, fit the data to
the most appropriate model.
3. A similarity region is set based on variation of parameters of the
fitted model for test units (e.g., capsules or tablets) from the
standard approved batches.
4. Calculate the MSD in model parameters between test and reference
batches.

5. Estimate the 90% confidence region of the true difference between

the two batches.
6. Compare the limits of the confidence region with the similarity
region. If the confidence region is within the limits of the similarity
region, the test batch is considered to have a dissolution profile
similar to that of the reference batch [7].
WAIVERS BASED ON IN VIVO-IN VITRO CORRELATION
For modified-release formulations, it is possible to obtain in vivo

bioavailability/bioequivalence waivers based on in vitro dissolution for
changes in formulations that usually require an in vivo study. The IVIVC
guidance released by the FDA in September 1997 [9] recommends that in
vivo bioequivalence studies for extended release products could be waived
if the sponsor has developed a correlation whose predictability has been
evaluated. In most cases, a level A correlation whose predictability has
been properly evaluated is used to establish the usefulness of the in vitro
dissolution as a surrogate for the bioavailability of the product under
question. In this case, the waiver is granted if the difference in the predicted
mean CMAX and AUC between the test and reference product is not more
than 20%.
If an IVIVC is developed with the highest strength, waivers for changes
made on the highest strength and any lower strengths may be granted if
these strengths are compositionally proportional or qualitatively the same,
the in vitro dissolution profiles of all the strengths are similar, and all strengths
have the same release mechanism.
This biowaiver is applicable to new strengths lower than the highest
strength, within the dosing range that has been established to be safe and
effective, if the new strengths are compositionally proportional; have the
same release mechanism; have similar in vitro dissolution profiles; and are
manufactured using the same type of equipment and the same process at the
same site as other strengths that have bioavailability data available.
For generic products to qualify for this biowaiver, one of the following
situations should exist:
– Bioequivalence has been established for all strengths of the

reference-listed product.
– Dose proportionality has been established for the reference-listed
product, and all reference product strengths are compositionally
proportional or qualitatively the same, have the same release
mechanism, and the in vitro dissolution profiles of all strengths
are similar.

462 Marroum et al.
– Bioequivalence is established between the generic product and

the reference-listed product at the highest and lowest strengths
and, for the reference-listed product, all strengths are
compositionally proportional or qualitatively the same, have the
same release mechanism, and the in vitro dissolution profiles are
similar.
To obtain a waiver for establishing bioequivalence of a new strength for a
generic product, the difference in predicted means of CMAX and AUC should
be no more than 10% based on dissolution profiles of the highest strength
and lower strength product.
The IVIVC guidance defines the following situations where an in vivo
bioavailability/bioequivalence cannot be granted even in the presence of an
established IVIVC:
a. Approval of a new formulation of an approved ER drug

product when the new formulation has a different release
mechanism.
b. Approval of a dosage strength higher or lower than the doses
that have been shown to be safe and effective in clinical trials.
c. Approval of another sponsor’s MR product even with the same
release-controlling mechanism.
d. Approval of a formulation change involving a nonrelease-
controlling excipient in the drug product that may significantly
affect drug absorption. For more detailed information on the
development, evaluation and applications of IVIVC, the reader is
reffered to Chapter 18 on this topic.
WAIVER OF IN VIVO BIOEQUIVALENCE BASED ON

BIOPHARMACEUTICS CLASSIFICATION SYSTEM
Waiver considerations based on the BCS approach are currently applicable

to IR products only. Also, BCS-based biowaivers are not applicable to
“Narrow Therapeutic Range” drugs and products designed to be absorbed
in the oral cavity [10].
The BCS is a scientific framework for classifying drug substances based
on two fundamental properties of a drug substance, i.e., its aqueous
solubility and intestinal permeability. A drug substance can have either a
high- or a low-aqueous solubility and either a high- or a low-intestinal
permeability. Thus, there are four BCS classes: Class 1 (High Solubility-
High Permeability); Class 2 (Low Solubility-High Permeability); Class 3
(High Solubility-Low Permeability); and Class 4 (Low Solubility-Low
Permeability). In addition, BCS also takes into account drug product

dissolution, and a drug product can have either a rapid or slow dissolution.
Thus, the BCS takes into account three major factors that govern the rate
and extent of drug absorption from IR solid oral dosage forms: dissolution,
solubility, and intestinal permeability. The central principle behind BCS-
based biowaiver considerations is that when the in vivo dissolution of an
IR solid oral dosage form is rapid in relation to gastric emptying and the
drug has high permeability, the rate and extent of drug absorption is
unlikely to be dependent on drug dissolution and/or gastrointestinal transit
time. Under such circumstances, demonstration of in vivo BA or BE may
not be necessary for drug products containing Class 1 drug substances
that exhibit rapid in vitro dissolution, as long as the inactive ingredients
used in the dosage form do not significantly affect absorption of the active
ingredients.
For BCS-based waiver considerations, the drug substance should be highly
soluble and highly permeable and the drug product should be rapidly
dissolving. Each of these criteria is defined further below.
Solubility. The solubility class boundary is based on the highest dose
strength of an IR product that is the subject of a biowaiver request. A drug
substance is considered highly soluble when the highest dose strength is soluble
in 250 mL or less of aqueous media over the pH range of 1–7.5.
Permeability. The permeability class boundary is based indirectly on the
extent of absorption (fraction of dose absorbed, not systemic BA) of a drug
substance in humans and directly on measurements of the rate of mass transfer
across human intestinal membrane. Alternatively, nonhuman systems capable
of predicting the extent of drug absorption in humans can be used (e.g., in
vitro epithelial cell culture methods). In the absence of evidence suggesting
instability in the gastrointestinal tract, a drug substance is considered to be
highly permeable when the extent of absorption in humans is determined to
be 90% or more of an administered dose based on mass balance determination
or in comparison to an intravenous reference dose.
Dissolution: An IR product is considered rapidly dissolving when no less
than 85% of the labeled amount of the drug substance dissolves within 30
minutes, using U.S. Pharmacopeia Apparatus I at 100 rpm (or Apparatus II
at 50 rpm) in a volume of 900 mL or less in each of the following media: (1)
0.1 N HC1 or Simulated Gastric Fluid USP without enzymes; (2) at pH 4.5
buffer; and (3) a pH 6.8 buffer or Simulated Intestinal Fluid USP without
enzymes. A sponsor/applicant can request waiver of in vivo BA and/or BE
studies for IR solid dosage forms based on BCS approach during the IND,
NDA, ANDA, and supplemental stages of an application. These waivers are
intended to apply to subsequent in vivo BA or BE studies after initial
establishment of the in vivo BA of IR dosage forms during the IND period,
and in vivo BE studies of IR dosage forms in ANDAs and postapproval
period.

464 Marroum et al.
Once the in vivo BA of a formulation is established during the IND period,

waivers of subsequent in vivo BE studies, following major changes in
components, composition, and/or method of manufacture (e.g., similar to
SUPAC-IR Level 3 changes) may be possible using the BCS. Biopharmaceutics
classification system-based biowaivers are applicable to the to-bemarketed
formulation when changes in components, composition, and/or method of
manufacture occur to the clinical trial formulation, as long as the dosage
forms have rapid and similar in vitro dissolution profiles. This approach is
useful only when the drug substance is highly soluble and highly permeable
(BCS class 1), and the formulations pre- and post-change are pharmaceutical
equivalents. Biopharmaceutics classification system-based biowaivers are
intended only for BE studies. They do not apply to food-effect BA studies or
other pharmacokinetic studies.
For ANDAs, BCS-based biowaivers can be requested for rapidly dissolving
IR products containing highly soluble and highly permeable drug substances,
provided that the reference-listed drug product is also rapidly dissolving and
the test product exhibits similar dissolution profiles to the reference-listed
drug product. This approach is useful when the test and reference dosage
forms are pharmaceutical equivalents. The choice of dissolution apparatus
(USP Apparatus I or II) should be the same as that established for the reference-
listed product.
Biopharmaceutics classification system-based biowaivers can be
requested for significant postapproval changes (e.g., Level 3 changes in
components and composition) to a rapidly dissolving IR product
containing a highly soluble, highly permeable drug substance, provided
that dissolution remains rapid for the postchange product and both pre
and postchange products exhibit similar dissolution profiles. This
approach is useful only when the drug products pre and postchange are
pharmaceutical equivalents.
For additional details like methodology, etc., the reader is referred to
the guidance [10]. It should also be noted that there is a great amount of
research and activity currently going on in terms of whether it is possible
to extend the limits of criteria by which a drug can be classified as BCS
class 1 as well as whether BCS-based waivers can be extended into other
BCS classes, and the reader is encouraged to keep abreast of peer-reviewed
journals in the area of biopharmaceutics as the debate and discussion on
BCS continues!

JAPANESE GUIDELINES FOR IN VIVO BIOAVAILABILITY/

BIOEQUIVALENCE WAIVERS
On February 14th 2000, the Japanese regulatory health agency issued two
guidances, the first entitled: “Guideline for bioequivalence studies for
formulation changes of oral solid dosage forms” [11], the second entitled
“Guideline for bioequivalence studies for different strengths of oral solid
dosage forms” [12]. These guidelines define the levels of changes in individual
excipients and categorize them into five different levels which are summarized
in Table 3 and 4. When the ratios of compositions are identical between test
and reference products, the formulation change is level A. This means that
test and reference products are the same in ratios of all components including
coating agents and, in the case of coated products, the weight of film and/or
sugar-coated layers per surface area of the core must be the same. When the
ratios are not identical, the levels of changes in individual excipients and
categorized excipients in Tables 3 and 4 should be determined. If the change
is equal to or less than the ranges of level B, it is level B. If the change is more
than the ranges of level B and equal to or less than the ranges of level C, it is
level C. Similarly, the change in excipients in the range between C and D is
TABLE 3 Level of Change in Individual and Categorized Excipients

(Uncoated Product)
Figures show the percent excipient (w/w) compared to total dosage form weight.
1
E.g., preservatives, stabilizer. Excipients of trace use are excluded.
2
Total additive effects of all excipient changes.

466 Marroum et al.
TABLE 4 Test Requirements for Each Level of Change as a Function of Therapeutic

Range and Solubility for IR, DR, and CR Dosage Forms
1
IR, DR, and CR mean immediate release (conventional), delayed-release (enteric
coated) and controlled-release dosage forms, respectively.
2
Products containing low solubility drugs are determined by dissolution tests. When
dissolution from the reference product does not reach 85% at 2 hr at pH 1.2 and 6 hr
at other pHs by paddle method at 50 rpm without surfactants, the drug is low solubility.
3
Single and multiple dissolution tests mean the test performed under specification
conditions and those under multiple conditions. When equivalence in dissolution is
not shown, in vivo tests should be performed according to the guideline for
bioequivalence studies of generic products.
level D. Any change in excipients whose use is limited to a trace is level A.

Among the above changes, the highest level of change is defined as the level
of formulation change. In the case of enteric coated products, the change in
the size of the dosage form from less than 4 mm to more than 4mm or vice
versa is a formulation change of level E. Depending on the level of change
and the comparability of dissolution profiles in one or more dissolution
medium, a bioequivalence waiver could be granted. Table 5 summarizes the
regulatory requirements for each level of change.
When multiple dissolution tests are recommended or in situations where
there is no approved dissolution method, the following is a description of
the required dissolution tests:
Dissolution tests should be performed, using a suitably validated
dissolution system and assay according to the following conditions:
1. Number of units: 12 units or more under each testing condition.
2. Testing time: 2hr in pH 1.2 medium and 6hr in other test fluids.
The test can be stopped at the time when the average dissolution of reference
product reaches 85%.

TABLE 5 Level of Change in Individual and Categorized Excipients (Coated Product)
Figures show percent excipient (w/w) compared to total dosage form weight. 1E.g.,
preservatives, stabilizer. Excipients of trace use are excluded.
2
Total additive effects of all excipient changes
3
Except for sugar-coated layer, all film-coated layers for water-proofing, undercoating,
enteric coating, and controlled-release are included.
4
Excipients of trace use are excluded
5
The surfaces of cores are determined from the shapes of dosage forms. If it is difficult,
the surface should be calculated under the assumption that the cores are spheres and
the densities do not change with the formulation change.
3. Testing conditions: The test should be carried out under the following
conditions.
Apparatus: JP paddle apparatus.
Volume of test solution: Usually 900 mL.
Temperature: 37°+/-0.5.

468 Marroum et al.
Test solutions: The 1st and 2nd fluids for the disintegration test (JP13)
are used as pH 1.2 and 6.8 test solutions, respectively. Diluted McIlvaine
buffers (0.05 M disodium hydrogen phosphate/0.025 M citric acid) are used
for other pH solutions. Other suitable test fluids can be employed when the
average dissolution of the reference product does not reach 85% at 6hr in
the McIlvaine buffers.
Products Containing Acidic Drugs
The test solution should be selected which provides the slowest dissolution
from the reference product and gives an average of 85% dissolution or more
within the testing time specified, 2hr at pH 1.2 and 6hr at other pHs. If the
dissolution from the reference product does not reach 85% at the specified
time in any test fluids, the test solution providing the fastest dissolution
should be used.
Products Containing Neutral or Basic Drugs, and Coated

Products
The test solution should be selected which provides the slowest dissolution
from the reference product and gives an average of 85% dissolution or more
within the testing time specified, 2hr at pH 1.2 and 6hr at other pHs. If the
dissolution from the reference product does not reach 85% at the specified
time in any test fluids, the test solution providing the fastest dissolution
should be used.

Products Containing Low Solubility Drugs

When the average dissolution from reference product does not reach 85% at
the testing time specified (2hr at pH 1.2 and 6hr at other pHs) at 50rpm in
any of the test fluids, without surfactants, employed in the above dissolution
tests (1) and (2), they are defined as products containing low solubility drugs.
Among 0.01, 0.1, 0.5, and 1.0 w/w% of polysorbate 80, the lowest
surfactant concentration should be chosen, which provides an average of
85% dissolution or more at the testing time specified (2 hr at pH 1.2 and 6
hr at other pHs) in at least, one of the test fluids. Dissolution tests in the four
fluids should be performed at the same surfactant concentration chosen. If
the average dissolution from the reference product does not reach 85% at
the specified time in any of test fluids, the surfactant concentration providing
the fastest dissolution should be selected. Among the three test solutions, the
testing fluid providing the slowest dissolution from the reference product
and giving an average 85% dissolution or more within the testing time
specified should be selected. If the average dissolution from the reference
product does not reach 85% at the specified time in any of the test fluids, the
test solution providing the fastest dissolution should be used.
Enteric Coated Products

470 Marroum et al.
Enteric coated products containing low solubility drugs should be tested by

adding polysorbate 80 to the test fluids (2) and (3) according to the dissolution
test for products containing low solubility drugs as described above.
Acceptance Criteria for Equivalence of Dissolution Profiles

The acceptance criteria for equivalence of dissolution profiles is based both
on average and individual dissolution profiles. Test and reference products
are considered equivalent when they meet both requirements (1) and (2)
shown below. The rule is not applicable to conventional dosage forms and
enteric coated products, unless the average dissolution from the reference
product reaches 85% under any of the testing conditions. If a dissolution
lag is observed for reference products, the equivalence in dissolution can
be assessed using the dissolution profile normalized for the lag time (see
below).
Average Dissolution
a. When the average dissolution from the reference product reaches 85%
within 15min: The average dissolution from the test product also reaches
85% within 15min or does not deviate by more than 10% from that of the
reference product at 15min.
b. When the average dissolution from the reference product reaches 85%
between 15 and 30min: The average dissolution from the test product does
not deviate by more than 10% from that of the reference product at two
time points where the average amounts dissolved from the reference product
are around 60 and 85%. When f2 is used, the f2 value should not be less than
50.
c. When the average dissolution from the reference product does not reach
85% in 30min: The following criteria should be applied to the comparison
of average dissolution profiles (2hr at pH 1.2 and 6hr at other pHs for
conventional and enteric coated products and 24 hr for controlled-release
products).
When the dissolution profiles are normalized for the lag time, the difference
in average lag time between test and reference products should not be more
than 10min.
d. When the average dissolution from the reference product does not reach
50% at the testing time point: The average dissolution of test product does
not deviate by more than 6% from that of the reference product at the time
points specified, or the f2 value is equal to or more than 60.
When the average dissolution from the reference product is between 50
and 85% at the testing time point:
The average dissolution of the test product does not deviate by more than

8% from that of the reference product at the time points specified, or the f2
value is equal to or more than 55.
e. When the average dissolution from the reference product reaches 85%
within the testing time: the average dissolution from the test product does
not deviate by more than 10% from that of the reference product at the time
points specified, or the f2 value is equal to or more than 50.
Individual Dissolution
Test products (n=12) should meet one of the following requirements at the
final time points where the average dissolution is compared between test
and reference products.
a. When the average dissolution of the reference product does not reach
50% within the testing time: There is no sample of test products that shows
the deviation of more than 15% in dissolution from the average dissolution
of the reference product, and one or no sample that shows the deviation of
more than 10%.
b. When the average dissolution of the reference product is between 50
and 85% at the testing time point: There is no sample of test product that
shows a deviation of more than 20% in dissolution from the average
dissolution of the reference product, and one or no sample that shows a
deviation of more than 12%.
c. When the average dissolution of the reference product reaches 85%
within the testing time: There is no sample of test product that shows a
deviation of more than 25% in dissolution from the average dissolution of
the reference product, and one or no sample that shows a deviation of more
than 15%.
Time Point for f2

a. When the average dissolution from the reference product reaches 85%
between 15 and 30min: 15, 30, and 45min.
b. When the average dissolution from the reference product reaches 85%
between 30 min and the testing time point: Ta/4, 2Ta/4, 3Ta/4, and Ta, where
Ta is the time point at which average dissolution from the reference product
reaches approximately 85%.
c. When the average dissolution from the reference product does not reach
85% at the testing time point: Ta/4, 2Ta/4, 3Ta/4, and Ta, where Ta is the
time point at which average dissolution from the reference product reaches
approximately 85% of the final amount dissolved in the testing time.
When there is a lag in dissolution, dissolution data normalized for the lag
time should be used for the calculation of f2.

472 Marroum et al.
Normalization of Dissolution Profiles with Lag Time

The lag time is conventionally defined as the time when 5% of the drug
dissolves. The lag time should be determined for individual dissolution by
linear interpolation, followed by normalization of dissolution profiles for
the lag time. Then, the average dissolution profiles are determined which
can be used for the assessment of equivalence in average dissolution.
EUROPEAN GUIDANCE FOR AN IN VIVO BIOAVAILABILITY

According to the European Agency for the Evaluation of Medicinal Products

guidance on the investigation of bioavailability and bioequivalence [14] if a
new application concerns several strengths of the active substance, a
bioequivalence study investigating only one strength may be acceptable.
However, the choice of the strength used should be justified on analytical,
pharmacokinetic, and safety grounds. Furthermore, all of the following
conditions should be fulfilled:
– The pharmaceutical products are manufactured by the same

manufacturer and process.
– The drug input has been shown to be linear over the therapeutic
dose range (if this is not the case the strengths where the sensitivity
is largest to identify differences in the two products should be
listed).
– The qualitative composition of the different strengths is the same.
– The ratio between amounts of active substance and excipients is
the same, or, in the case of preparations containing a low
concentration of the active substance (less than 5%), the ratio
between the amounts of excipients is similar.
– The dissolution profile should be similar under identical conditions
for the additional strengths and the strength of the batch used in
the bioequivalency study.
If a new strength (within the approved dose range) is applied for on the basis
of an already-approved medicinal product and all of the stated conditions
hold then a bioequivalence study is not necessary.
In case of exemption from bioequivalence studies, in vitro data should
demonstrate the similarity of dissolution profile between the test product
and the reference product in each of the three buffers within the range of pH
1–8 at 37°C (preferably at or about pH 1, 4.6, and 6.8). This is done using
the f2 similarity factor. However, in cases where more than 85% of the active

substance is dissolved within 15 min, the similarity of dissolution profiles

may be accepted without any mathematical evaluation.
CANADIAN GUIDANCE FOR IN VIVO BIOAVAILABILITY/

According to the Drug Directorate of Canada guideline on the conduct and

analysis of bioavailability and bioequivalence studies for uncomplicated drugs
in which the proportions of excipients to the drug and the dissolution
characteristics are the same, it is sufficient to establish the bioavailability of
one strength. Whether all strengths of other products should be tested will
depend on the extent to which the other formulations differ in strength. For
some of the complicated drugs such as those with narrow therapeutic range,
steep dose response characteristics, or nonlinear kinetics, a single-dose
bioavailability study should be conducted on each strength [15].
CONCLUSION
More and more regulatory agencies around the world are relying on in vitro
dissolution to assess the bioavailability and the bioequivalence of drug
products. The dissolution test is no longer looked at as only a quality control
tool but also as an indicator of the bioavailability of a drug product. Minor
formulation changes can be approved just based on dissolution data and
even major formulation changes that required bioequivalence studies in the
past are being waived if the drug belonges to BCS class I or if there is a
predictive IVIVC. Thus dissolution testing if done properly can result in
decreasing the regulatory burden on sponsors by decreasing the number of
in vivo studies that are needed to approve and maintain a drug product on
the market. That is why during the development stage of a drug, proper care
and attention should be paid to develop the most appropriate dissolution
method that is discriminatory and that will as much as possible have the
ability to reject formulations or lots with an inadequate in vivo bioavailability
profile.
REFERENCES
1. Code of Federal Regulations 21 321.10.

3. Guidance on Bioavailability and Bioequivalence Studies for Orally Administered
Drug Products—General Considerations Center for Drug Evaluation and
Research, Food and Drug Administration, October 2000.

474 Marroum et al.
4. Marroum, P.J. Bioavailability/Bioequivalence for Oral Controlled Release

Products, Controlled Release Drug Delivery Systems: Scientific and Regulatory
Issues, Fifth International Symposium on Drug Development, East Brunswick,
NJ, May 15–17, 1997.
5. Guidance for Immediate Release Solid Oral Dosage Forms, Scale Up and Post
Approval Changes:Chemistry and Controls: In Vitro Dissolution testing and In
Vivo Bioequivalence Documentation, Center for Drug Evaluation and Research,
Food and Drug Administration, November 1995.
6. Approved Drug Products with Therapeutic Equivalence, 20th Ed.; vii–viii, Center
for Drug Evaluation and Research, Food and Drug Administration, 2000.
7. Moore, J.W.; Planner, H.H. Mathematical Comparison of Dissolution Profiles.
Pharmaceutical Technology 1996, 6, 64–74.
8. Guidance on Dissolution Testing of Immediate Release Solid Oral Dosage Forms,
Center for Drug Evaluation and Research, Food and Drug Administration, August
1997.
10. Guidance for Modified Release Solid Oral Dosage Forms, Scale Up and Post
Approval Changes: Chemistry and Controls: In Vitro Dissolution testing and In
Vivo Bioequivalence Documentation, Center for Drug Evaluation and Research,
Food and Drug Administration, October 1997.
11. Guidance on Extended Release Dosage Forms: Development, Evaluation and
Aplications of In Vitro In Vivo Correlations, Center for Drug Evaluation and
Research, Food and Drug Administration, September 1997.
12. Guidance on Waivers of In Vivo Bioavailability and Bioequivalence Studies for
Immediate Release Solid Oral Dosage forms based on Biopharmaceutics
Classification System, Center for Drug Evaluation and Research, Food and Drug
Administration, August 2000.
13. Guideline for Bioequivalence Studies for Formulation Changes of Oral Solid
Dosage Forms, Japanese National Institute of Health Sciences, February 2000.
14. Guideline for Bioequivalence Studies for Different Strengths of Oral Solid Dosage
Forms, Japanese National Institute of Health Sciences, February 2000.
15. Guideline for Bioequivalence Studies of Generic Products, Japanese National
Institute of Health Sciences.
16. Note for Guidance on the Investigation of Bioavailability and Bioequivalence,
The European Agency for the Evaluation of Medicinal Products, July 2001.
17. Guideline on the Conduct and Analysis of Bioavailability and Bioequivalence
Studies—Part B: Oral Modified Release Formulations, Therapeutic Products
Programme Health Canada.

20
Bioavailability and Bioequivalence

Issues for Drugs Administered via
Different Routes of Administration;
Inhalation/Nasal Products;
Dermatological Products, Suppositories
Edward D.Bashaw
Rockville, Maryland, U.S.A
OVERVIEW
While oral dosage forms represent the preferred route of drug delivery, there
are situations when nonoral routes are indicated. In this chapter, we will
present an overview of the issues involved in assessing bioavailability and
bioequivalence via nonoral routes of administration. Each route of
administration will be presented individually along with a discussion of some
of the pharmaceutic and physiologic factors affecting drug absorption.
Examples of how some of these factors can interplay in the design and
evaluation of these dosage forms will also be presented.
INTRODUCTION
While oral dosage forms are the primary route of delivery of most
Pharmaceuticals there are times where either due to pharmacokinetic factors
475
476 Bashaw
(such as first-pass metabolism), or due to a desire to minimize systemic effects

through local administration, the disease state itself (i.e., extreme nausea
and vomiting) will not allow for oral dosing. In these situations, alternative
routes of administration must be utilized to obtain the desired therapeutic
outcome. Consequently, in the development of drugs for these routes of
delivery great care must be taken to consider the unique challenges that each
of these routes presents in relation to bioavailability and bioequivalence
testing.
The nasal, dermatological, and rectal routes of administration, although
on the surface are quite distinct, they are all linked in that they are all, in a
broad sense, examples of topical drug application but not necessarily topical
drug delivery. The difference is that in topical drug delivery the drug is
administered for a local effect, as is most often the case in applying drugs to
the skin. In comparison, both the intranasal and rectal routes are often chosen
to provide systemic drug delivery under special circumstances.
Inherent in these three routes of administration is the fact that all of them
are not normally thought of as being naturally permeable to drug absorption
to any great extent. For example, the skin is first and foremost a barrier
protecting internal tissues from external insult, be they chemical, bacterial,
or physical in nature. Likewise, in the nose, the nasal passages and structural
components are present not for drug absorption but to act as a filter to
remove inhaled pollen, bacteria, and other suspended particulates prior to
their delivery to the lung. The rectum, while the distal end of the digestive
tract, does not have the structure of the small intestine or the enzymes and
digestive juices present to enhance nutrient absorption. Because none of these
tissues are inherently designed for drug/nutrient absorption choosing them
as a site for drug delivery requires an assessment of physiochemical properties
of the drug, the target tissues, and the performance mechanics of the drug
delivery device/vehicle.
INHALATION/NASAL DRUG PRODUCTS
For the most part, the application of drug substances to the nasal mucosa
has historically been limited to topically acting agents for the symptomatic
treatment of allergic rhinitis and the common cold. In the last ten to fifteen
years a renewed interested in the nasal route of drug delivery has occurred as
a method of delivering protein-based therapeutic agents that would be
unstable in the gastric/digestive environment. The archetypical drug that has
been proposed in the literature is insulin. Insulin given by the intranasal
route would provide a quicker onset of action, relative to subcutaneous use,
and would be more physiologic in its action. The delivery characteristics of
insulin and other small protein-based drug products such as vaccines via the

Bioavailability and Bioequivalence Issues 477
intranasal route are a route of great promise for the small bioactive molecules
and are actively being pursued.
Anatomy and Physisology of the Nose

Although the external nasal tissue differs markedly in size and shape from
individual to individual, regardless of its external size, it is the large internal
surface area of the nose, which helps it perform its many functions as both
sensory and respiratory organ (Fig. 1). While the shape of the external nasal
tissue, the “nose” itself, may in severe instances restrict airflow, this does not
routinely play a role in the delivery of drug to the nasal tissues due to
placement of the pump/spray unit within the nasal cavity. The barriers to
drug absorption in the nose can be classified as mechanical (cilia function),
passive entrapment (mucous production), and enzymatic (nasal P-450
activity).
As a filter, the nasal mucosa prevents the entry of particles larger than 5
µm in diameter, and most smaller particles into the lower respiratory tract.
FIGURE 1 The internal surface area of the nose. Source: Ref. 60, p. 312.

478 Bashaw
In addition, the nose through its extensive vascular supply rapidly, but
only partially, regulates the temperature and humidity of inhaled air (~
10,000 L per day) despite changes in external air temperature that can
rapidly change from a heated room to subzero conditions. The ability of
the nose to filter particles efficiently from inspired air is accomplished by
several mechanisms. A large proportion of inhaled particulate matter is
deposited at the anterior unciliated area of the nasal passages as a direct
result of filtration by nasal vestibule (i.e., the external nasal tissues). The
nasal valve at the posterior end of the vestibule limits the rate of inspiratory
nasal air flow and accounts for ~50% of the total resistance to airflow
from ambient air to the alveoli. Internally, the nasal turbinates increase the
mucosal surface area of the nasal cavity to approximately 100 to 200cm2
and regulate airflow by changing the blood content of the highly vascular
turbinates both spontaneously and rhythmically (i.e., the “nasal cycle”).
The turbulence of the air passing through the nose also helps cause
impaction of particles and assists all the other functions of the nose. These
cyclic changes in resistance to airflow occur in 80% of normal subjects;
each nasal cycle lasts from two to six hours. As airway resistance increases
in one nostril, it decreases in the other.
Inspired particles are further filtered by their entrapment of inhaled
particles in a mucous “blanket”, on the surface of the ciliary epithelium
approximately 10 to 15 µm deep. This mucous “blanket” starts posterior to
the anterior tip of the inferior nasal turbinate and covers the entire nasal
cavity. It is a watery mixture consisting primarily of proteins, six of which
are derived from plasma. The mucus is secreted by surface goblet cells that
line the nasal cavity. The principal protein and antibody present is
immunoglobulin A (IgA), which is synthesized against viral respiratory
infection antigens as well as other antigens. Besides this antigen antibody
response the mucous provides a physical barrier and effectively traps and
removes particles greater than 4µm in diameter. Mucociliary transport moves
the blanket, with its contents, posteriorly toward the nasopharynx at an
average rate of 8 to 9 mL per min, except at the anterior portion of the
inferior turbinates where it moves anteriorly. Throughout the day the normal
pH of the nasal cavity varies between 5.97 and 7.85 and is markedly constant
showing no change in response to rest or meals. Ideally, it is into this milieu
that inspired drug particles are trapped and become solubilized for delivery
to the nasal tissues for absorption. In contrast drug particles that become
directly lodged in the cilia are rapidly cleared under normal circumstances.
Environmental irritants such as tobacco smoke may significantly decrease
the ciliary activity of the nasal mucosa. If destroyed as a result of infection,
the epithelium can regenerate, although such regeneration may take from a
few hours to two weeks postinfection depending on the depth and scope of
the insult. Occasionally, following either a massive acute insult or the result

of a chronic disease process, the nasal mucosa does not regenerate. In such
cases, the filtering ability of the nose is greatly decreased and larger particles
are allowed to penetrate deeper into the respiratory tract. Normally clearance
of particles from the nasal mucosa occurs within 15min of deposition by the
combined effects of mucous trapping and ciliary action. This “residence time”
in the nasal mucosa can be increased into hours by the in situ formation of a
bioadhesive or “mucoadhesive” delivery system, allowing for localization of
drug and enhancement of drug delivery. This has implications for drug delivery
where particle size control of droplet formation is critical to targeted drug
delivery.
Drugs for Nasal Delivery

As mentioned previously the primary use of drugs administered intranasally
has been to treat allergic rhinitis and the common cold. This includes agents
such as the topical corticosteroid (betamethasone, fluticasone, budesonide,
etc.) topical vasoconstrictors (oxymetazoline, phenylephrine, etc.) and other
miscellaneous agents such as cromolyn sodium. All of these agents work to
improve airflow from the nasal mucosa by either dilating the nasal passages
(Fig. 1) or decreasing the immune response via local mechanisms of action.
As such bioavailability/bioequivalence testing of such compounds is limited
by the small doses administered and the biological response.
Systemic drugs such as intranasal butorphanol (Stadol NS) and nicotine
(Nicotrol nasal spray) are delivered intranasally to either avoid first-pass
metabolism or provide effective drug levels rapidly. In the case of nicotine,
comparative in vivo bioavailability studies comparing the intranasal spray
to other routes of administration clearly shows that it produces plasma levels
inferior to those of a cigarette, but superior in rate to most other routes of
administration. Thus the intranasal spray form of nicotine provides rapid
vascular access to the brain without coadministration of the accompanying
carcinogens formed from the burning of tobacco. Used as a part of a smoking
cessation program the nasal spray can be effective in lessening and then
elimination of the addiction.
Bioavailability/Bioequivalence Considerations
General Considerations
For both systemically delivered agents and agents for topical treatment, the
following table summarizes some of the considerations which must be taken
into consideration in the design of a nasal dosage form and its proper
evaluation.

480 Bashaw
TABLE 1 Factors for Consideration in Designing a Nasal Dosage

Form
Inspection of this list reveals that many of these issues relate to the
development of the dosage form itself, i.e., chemistry and manufacturing
considerations rather than drug absorption. Only in nasal or inhalational
drug delivery does the delivery system itself play such a key role in the
biopharmaceutics of a drug. This is because with inhalational drug delivery
we are dispersing drug into the nasal passageways as suspended particles or
droplets that then must settle out in the appropriate location, relative to the
various elimination mechanisms present in the nasal cavity, for absorption
to become possible. In the assessment of nasal bioavailability/bioequivalence,
we must first consider whether or not the drug is intended for systemic or
topical action.
Topically Acting Drugs
With topically acting drugs, such as vasoconstrictors, in vivo determination

of systemic plasma levels is often impossible due to analytical constraints. In
such situations, use of pharmacodynamic endpoints such vital capacity and
forced expiration volume (FEV1) can be used as a surrogate measure of
bioavailability. In the case of corticosteroids, the assessment of the
hypothalamic-pituitary adrenal (HPA) axis suppression has been used as a
systemic marker of bioavailability/bioequivalence, even though the intended

site of action is local. In this latter case, it is the absence of effect on the HPA
axis that is demonstrative of localization of drug delivery to nasal tissues.
Prior to accepting such data for a new chemical entity or even a known
substance in a new formulation, an attempt should be made to first quantify
the in vivo plasma levels under maximal dosing conditions. With maximal
dosing conditions being defined as multiple dosing at the highest clinically
tested dose and dosing frequency. This is necessary as with new agents their
degree of absorption cannot be determined reliably by animal extrapolation,
and in the case of older known agents, developments in both delivery system
technology and analytical methodology may have reached the point of
producing systemic levels. Such in vivo pharmacokinetic trials need not
incorporate a large number of blood samples under the concept of a
surveillance pharmacokinetics sampling strategy. This sampling strategy
differs from standard geometric sampling in that it focuses the samples in
the time period within which blood levels would likely occur. That is to say,
with nasal products, given the mucocilliary elimination mechanisms present,
drug absorption from the nasal mucosa, from immediate release products,
beyond two to three hours is highly unlikely. Under a geometric sampling
strategy, which would space blood samples throughout the dosing interval,
numerous blood samples would essentially be wasted, adding to the
inconvenience of the subject and cost of the trial. By taking blood samples
only during those time periods when absorption would be expected to occur,
one can reduce both the inconvenience and the cost of the trial. The down
side to surveillance pharmacokinetics is that if significant and prolonged
drug levels are seen, the sampling strategy may not be sufficient to determine
the underlying pharmacokinetic systems. In practice, incorporating surveil-
lance pharmacokinetic sampling into an early phase II trial can minimize
this potential with a limited number of subjects. In either case, given the
recent advances an analytical technology over the last decade, more and
more agents that have in vivo pharmacodynamic/clinical efficacy assess-ments
for bioavilability testing will be replaced with in vivo pharmacokinetic
methods.
Systemically Active Agents

For those agents administered via the intranasal route for systemic effects
the performance/absorption of drug from the intranasal route should be
compared to that from another route of administration, be it oral or ideally
intravenous. Figure 2 shows the comparative in vivo bioavailability of
transnasal butorphanol relative to IV and sublingual administration, while
Fig. 3 shows the comparison of the nicotine nasal spray to other routes
including cigarettes. From both of these examples, the rapid nature of
intranasal absorption can be seen. In the case of butorphanol, its use as a

482 Bashaw
FIGURE 2 In vivo bioavailability of transnasal butorphanol relative to IV and sublingual

administration. Source: Ref. 21, p. 376.
FIGURE 3 Comparison of the nicotine nasal spray to other routes including cigarettes.
Source: Ref. 19, p. 76.

treatment for the pain of migraine headache would require a rapid onset of
action, compared to the IV formulation, the nasal spray has an absolute
bioavailability of ~50%. With this information proper dose-ranging and
treatment regimens can be designed and tested to maximize the attainment
of effective levels for analgesia.
As for the nicotine nasal spray, Fig. 3 shows that across a number of
different studies only the “gel” and nasal spray dosage forms show a rapid
increase in venous levels of nicotine compared to the gum or vapor form (an
early of the nicotine inhaler). Arterial levels of nicotine (Fig. 4) show that the
nasal spray can achieve arterial levels rapidly and thus respond more readily
to nicotine “craving” by subjects needing the rapid “hit” associated with
cigarettes that is lacking with the other formulations. By understanding the
need to provide quitting smokers with a nicotine delivery system that can,
albeit at a reduced level, provide a cigarette like rush of nicotine levels, the
relatively low rates of smoking cessation using nicotine replacement products
may be increased by responding to the needs and pattern of addiction and
addictive behavior.
FIGURE 4 Arterial versus venous levels of nicotine over time. Source: Ref. 7, p. 641.

484 Bashaw
From a formulation design aspect, studies should also be undertaken

to assess the impact of multiple actuation on bioavailability. In vitro and
in vivo studies have shown that when an insufficient amount of time has
elapsed between actuations, the suspended drug particles in the nasal
tissues often coalesce into larger particles that are more easily cleared by
the nose. In doing so the resulting in vivo bioavailability of the drug can
drop relative to the administered dose. This can result in an urge in the
patient to increase the dose, resulting in a further loss of bioavailability
and can result in reports of patient dissatisfaction with the product—a
situation that proper study and patient counseling by the physician and
pharmacist can overcome.
Disease State
As these products are being administered for systemic effects, consideration
must be given to the impact of other disease states on drug absorption.
Specifically, the effect of allergic rhinitis with its attendant copious nasal
discharge should be evaluated along with the impact of topical vasocon-
strictors on drug absorption. In both the situations, the impact on drug
absorption needs to be determined so that the dose and or dosing
instructions can be altered to maintain effective in vivo plasma concentra-
tions. In Fig. 5, the results of a comparative in vivo bioavailability study in
which smokers were given the nicotine nasal spray both in the absence of a
cold and in the presence of a cold with xylometazoline. Clearly the peak
plasma levels are blunted and the time to achieve these plasma levels is
increased from a disease-free baseline of 0.28 to 0.40 hr with rhinitis alone,
and to 0.52hr with rhinitis/xylometazoline. In a situation like nicotine
replacement therapy or in the case of butorphanol, when pain relief is the
endpoint, the existence of rhinitis, with or without concomitant use of a
topical vasoconstrictor, can significantly affect the onset and quality of
drug effect. These factors need to be considered in drug development along
with strategies, for either dosing increases, or rescue/alternative treatment
regimens during the time course of the cold.
Structural defects in the nose, be it a deviated nasal septum or other
structural abnormality in the nasal passage can also affect the bioavailability
of nasally administered drugs. However, the wide variety and severity of
these defects are such that a systematic study of them prior to drug approval
is not feasible. Labeling should be developed with this in mind to instruct the
prescriber to consider this potentiality in selecting patients for intranasal
drug delivery.

FIGURE 5 Results of a comparative in vivo bioavailability study in which smokers

were given the nicotine nasal spray both in the absence of a cold and in the presence
of a cold with xylometazoline. Source: Ref. 14, p. 73.
Delivery System
Unique to the intranasal (and other inhalational routes of drug delivery) is
that additional studies may be required to assess the performance
characteristics of the delivery system itself. That is the reproducibility of the
pump/device to delivery a consistent dose from first to last, both in the amount
of drug delivered and the production of the proper-sized particles. When
possible, absolute in vivo bioavailability studies should be undertaken to
determine the efficiency of the interaction between the drug-formulationroute
of delivery factors previously outlined in Table 1. The data from such studies
should be used to optimize the formulation in terms of delivery by
modification of the particle size and spray pattern produced by the nozzle at
the point of delivery.
Dosing Instructions
Prior to the use of a nasal inhaler/spray device the subject should, in turn,
clear each nostril by blowing. In the case of rhinitis, the subject may wish to

486 Bashaw
use a topical vasoconstrictor 20–30 min prior to dosing. The inhaler/spray

device should be placed into the nose and with the contralateral side of the
nose occluded the dose should be delivered in time with a natural intake of
air. The breath should then be held for 15–20 seconds to allow time for the
drug particles to settle out and become available for absorption. Then the
contralateral side of the nose should be dosed according to directions, or in
the case where instructions are not given, two to five min after the first dose.
By providing a delay between doses, the potential for suspended droplets to
coalesce into larger particles, which are more readily eliminated, is minimized.
TOPICAL DRUG DELIVERY
Topical drug delivery differs from transdermal drug delivery in that the sites
of drug application and drug action are one and the same. In topical drug
delivery, we are primarily concerned with delivering drug to skin itself whereas
with transdermal drug delivery we are concerned with the delivery of drug
through the skin to the systemic circulation. For a topically applied agent,
drug that reaches the systemic circulation is essentially lost to the site of
action and can result in undesired side effects. Examples of such side effects
include suppression of the hypothalamic-pituitary adrenal (HPA) axis in the
case of topically applied corticosteroids or birth defects in the case of topical
retinoids. In this section, we will focus on the biopharmaceutic issues
surrounding topical drug application.
Anatomy and Physiology of the Skin

Prior to discussing the evaluation of topical drug delivery we must first
consider the skin and the various physiologic factors that affect it. The skin
is the largest organ in the body with a surface area in the adult male
approximating 1.73m2. It is a multifunction organ which besides its structural
role as a physical protective covering has important roles in thermoregulation
and maintaining fluid balance. It is the first line of defense against bacterial
infection and is undergoing continual replacement via the shedding of skin
cells.
The skin itself is organized into discrete layers that each have a role to
play in the structure and function of the skin. The outermost layer of the
skin is the stratum corneum. This layer, approximately 10–15 cells thick is
composed of dead skin cells (corneocytes) arranged in a so-called brick and
mortar pattern with lipids representing the mortar. It is devoid of blood
vessels and represents the primary barrier to the permeation of water and
drug delivery. In Fig. 6, the stratum corneum is shown as the outer layer of
the epidermis which can be further subdivided into the stratum granulosum

FIGURE 6 Layers of the epidermis. Source: Ref 60, p. 162.
and germinativum, and in the case of the thicker skin on soles of the feet and
hands, the stratum lucidum. The epidermis itself lacks a system of vascular
structures and is nourished by papillary capillaries in the dermis that extend
upward into finger-like projections of the dermis, called dermal papillare,
into the epidermis. In addition to the vascular supply for the epidermis, the
dermis also contains the elastin and collagen fibers that give skin its strength
and resilience along with sensory nerve fibers for pain, touch, and
temperature. Most topically treated diseases are thought to arise from the
upper stratum granulosum (i.e., fungal infection) to the dermis (i.e., atopic
dermatitis).
Systemic drug absorption following topical application can occur via a
number of mechanisms:
1. Direct absorption through the stratum corneum and epidermis

to the underlying capillaries,
2. Transfollicular drug delivery via the hair shaft.
3. Drug absorption through the eccrine (sweat) gland pathway.
Of these pathways, the transfollicular and eccrine glands represent potential

shunts of drug delivery that increase in importance, in normal skin, when
the stratum corneum is intact. In the setting of topical drug delivery, where
the stratum corneum is disrupted, these routes play a lesser role.
Transfollicular absorption can become a major route for absorption in those

488 Bashaw
situations where the site of drug action is the hair shaft itself. In the case of
pediculosis (lice), topical products are often formulated as a shampoo or
mousse to enhance the coating of the hair shaft. Drug is then carried down
to the follicle where it can be absorbed. Because of their lipophilic nature,
transfollicular absorption is thought to be a major route of pesticide
absorption in field workers.
Drugs for Topical Drug Delivery

As mentioned above topical drug delivery is designed to provide local
treatment to the skin and related tissues. This can be in response to a number
of diseases including acne vulgaris, actinic keratosis, atopic dermatitis,
psoriasis, fungal infection, and vertilligo to name but a few of many such
diseases. This represents a wide range of potential disease states and their
attendant treatments from antibiotics (erythromycin, clindamycin, etc.) for
acne, antifungals (terbinafine, ketoconazole, etc.) for athlete’s foot, retinoids
(retinoic acid, tazarotene, etc.) for psoriasis and corticosteroids
(betamethasone, clobetasol, etc.) for atopic dermatitis.
As in the case of intranasal drug delivery of locally acting drug products
the availability of a validated analytical method will determine the types of
in vivo bioavailability trials conducted. In addition to the drug, the vehicle
often plays an important part in the case of localizing drug to the target site.
Topical vehicles include creams, ointments, gels, solutions, lotions, mousse,
shampoos, foam, and variations on these themes. While it is tempting to
generalize that all lotions are more available than creams and ointments,
this is not always the case. With topically applied drugs absorption is
dependent on the interplay between the skin, vehicle, drug, and any
permeation enhancers that may be present in the formulation.
Bioavailability/Bioequivalence Considerations
General Design Factors
In most diseases of the skin, the structural layers and/or integrity of the skin
are disrupted, and drug penetration throughout the stratum corneum to the
other layers of the epidermis and dermis are altered. It is for this reason that
in vivo bioavailability studies should always be conducted in the target patient
population with disease severity approximating the upper limit of that allowed
for in the planned clinical development program. In this case, the use of
healthy normal volunteers is of no value in assessing the pharmacokinetics
of drug absorption in diseased skin. The only exception to this general rule
would be in the case of diseases of pigmentation (both hyper- and hypo-)
such as vitiligo in which case the underlying structure of the skin is unchanged.

In such situations as this, the use of normal subjects or areas of nondiseased

skin in subjects with the disease is allowable. However, individual study
guidance from the regulatory body in question should be sought to obtain
an agreement on this and other study design issues prior to study initiation.
Similar to the concepts used in the evaluation of intranasal dosage forms,
the underlying principle of pharmacokinetic study design in topical products
is to maximize the potential for systemic levels to occur. This is done by
modifying those factors that affect topical drug absorption, see Table 2.
By maximizing these elements in the setting of diseased skin one can often
produce systemic plasma levels, or in the case of corticosteroids, produce
clinically significant HP A axis suppression. Given the chronic nature of
many topical diseases, such as atopic dermatitis and psoriasis, systemic
availability and its assessment is critical to the overall safety determination
for a drug.
An example of the kind of plasma levels that can be achieved with topical
dosing under chronic conditions is that of tazarotene. When applied to normal
(i.e., non-diseased) skin, the systemic absorption of tazarotene is low (~1%)
even after multiple dosing. However, in three-month study of subjects with
psoriasis, with a mean total body area involvement of 13%, systemic levels
of tazarotenic acid (the active metabolite) were detectable with an estimated
bioavailability, upon multiple-dosing, of <5% (Fig. 7). Interestingly, with
continued-dosing, the bioavailability of tazarotene drops over time until it
approaches that of healthy individuals. This is thought to be due to the
retinoid effects on clearing the psoriatic plaques allowing the re-establishment
of an effective skin barrier, and thus decreasing the permeability of the skin
to tazarotene.
In vitro Methods
As mentioned earlier, when a sponsor is pursuing the development of multiple
topical formulations, an in vivo biostudy with the most bioavailable dosage
TABLE 2 Topical Bioavailability Study Design Elements

490 Bashaw
FIGURE 7 A three-month study of subjects with psoriasis, with a mean total body
area involvement of 13%, systemic levels of tazarotenic acid (the active metabolite)
were detectable with an estimated bioavailability, upon multipledosing, of <5%.
Source: Ref. 37, p. 280.
form may be sufficient under certain situations. Unfortunately, while in vitro

methods, using such apparatus as the Franz Diffusion Cell may be useful for
assessing the relative penetration of drug through intact skin. The relationship

between the degree of drug penetration of both diseased and normal skin
varies from disease to disease and within a disease according to severity and/
or extent of involvement. This basic alteration in skin structure severely limits
the utility of in vitro and novel in vivo test methods in the evaluation of
topical dosage forms. While they may be useful in the initial screening of
topical formulations in healthy adults or through the use of cadaver skin,
such methods as diffusion cells, tape stripping and microdialysis all share
these same limitations.
Age
Another element to be considered in the evaluation of these drugs is the age
of the patient population. Skin, like other organ systems ages and as it ages
it looses some of its structure and function including its ability to regulate
body heat and maintain fluid balance, see Table 3. Usually this is not a problem
in the performance of pharmacokinetic trials as it is usually much easier to
recruit older subjects than young children. In such situations where sufficient
numbers of subjects exist, a secondary pharmacokinetic analysis using both
gender and age as covariates should also be undertaken.
In contrast, in the pediatric population, especially in the neonate,
differences in skin maturation can be profound in relation to disease severity.
In adults the skin represents, on average, only 3% of total body weight while
in neonates it can go as high as 13%. This coupled with the fact that the
ratio of surface area to body weight in neonates is four times that of adults,
suggests that our relationships between surface area and volume need to
reconsidered. While term infants are born with and acquire all the
characteristics of an intact skin barrier, these high ratios of surface area to
weight would only tend to enhance the potential for circulating levels of
topically applied drugs to occur after application. In this situation,
extrapolation of in vivo biostudy results should be limited to that of younger
aged subjects to older subjects and not vice versa.
Because of the increased body weight to surface area ratio in children, the
absence of circulating plasma levels in children with the same relative degree
TABLE 3 Functions of Human Skin that Decline with Age

492 Bashaw
of surface area involvement, compared to adults, would be supportive of the

clinical safety findings across these populations. By the same token,
extrapolation of safety from adults to children is not possible for the same
reasons. In general, to obtain approval of a pediatric dosing regimen, one
has to study the age range in question with adequate numbers of subjects
being present at the lowest desired age ranges. While there is no hard and
fast rule as to the number of pediatric subjects required, the protocol should
prespecify the numbers of children at each age grouping (1–6 months, 6
months-2 years, 2–6 years, etc.). It should also indicate that the enrolled
children should be evenly distributed throughout the age range, if not enriched
at younger ages, to prevent clustering at the upper ages.
Dosing Instructions
Site preparation prior to the application of a topical product primarily consists
of washing the area of application with mild soap and water and patting dry.
Care should be taken to avoid the use of harsh soaps and detergent like
“liquid soaps” that would tend to strip out the natural oils present in the
skin and potentially alter drug delivery. The product should be applied,
according to directions, to the affected site, minimizing the exposure or
“normal” skin. Following application the subject should follow the specific
directions for the product concerning the use of a bandage or occlusive barrier
either of which could contribute to enhanced systemic absorption. As a general
rule the site should be allowed to air dry naturally following application,
before covering the area with clothes.
Special Situations—Minimal Surface Area Application

Across the spectrum of dermatologic conditions there are those conditions
like atopic dermatitis that can involve >90% of the total body surface area
and those that involve <1% or so of body surface area. Disease states in this
latter category include basal cell carcinoma and warts. These lesions are
usually circumscribed in nature being distinct from the surrounding skin
surface. Treatment of these lesions can include surgical removal and the use
of topically applied caustic agents (such as high concentrations of salicylic
acid or trichloroacetic acid). In these situations, where the destruction of
discrete and limited areas of skin are done, the utility of pharmacokinetic
monitoring is of questionable value for a number of reasons:
1. The small surface area involved
2. The destructive nature of the “drug” being applied to the lesion
3. The single use/application nature of these products.
In these situations, even the use of a minimal pharmacokinetic sampling
strategy becomes complicated, as one of the precepts of regulatory

decision-making is not to place the research subjects at any unnecessary

risk. While minimal, the act of drawing blood from a patient does carry
with it some risk. For these reasons, depending upon the ultimate surface
area to be treated at any one time and the total cumulative dose to be
applied at any one time, it may be possible to obtain a waiver of in vivo
bio-testing. Such considerations should be discussed with the regulatory
authorities early on in the development of a topical treatment for these
diseases and should not be assumed as a matter of course.
RECTAL DOSAGE FORMS
Since the early 1800s when cocoa-butter suppositories were first developed
by the French, use of the rectal route for drug administration has often been
proposed as an alternative method to avoid first-pass metabolism and as a
viable route of drug delivery in patients who cannot use oral dosage forms.
Today suppository dosage forms range from the original cocoa-butter
formulations, to those utilizing new polymers and dispersive systems
(including the use of oral controlled-release products) designed to overcome
one or the other problems associated with rectal administration. Even so,
the use of the suppository route in general, and the rectal route in particular
is one that is not often pursued in the course of modern drug development.
The only exception to this general statement is the proliferation in recent
years of antifungal and hormonal products in the form of vaginal
suppositories. Rectal suppositories, in comparison, are almost never developed
as a first route of administration and rarely as a line extension, except for
use in the infant or pediatric population.
Anatomy and Physiology of the Rectum

The rectum is the terminal end of the gastrointestinal (GI) tract. Its primary
function, different from any other portion of the GI tract, is not to absorb
nutrients or regulate fluid balance but to serve, in much the same way as the
bladder, as a holding place for waste materials prior to the regular daily
expulsion of these materials. The rectum is muscular in nature and does
have a high degree of vascularization, see Fig. 8.
One of the misconceptions regarding rectal drug delivery is that it bypasses
first-pass metabolism by avoiding the portal circulation. This is only partially
true. The superior, middle, and inferior rectal veins accomplish the removal
of blood from the rectal tissues. These veins are interconnected through
numerous anastamoses and as such represent a unified drainage system. Of
these three veins, the superior rectal vein does drain into the portal vein, thus
providing vascular access to the liver. Because of individual variability in the

494 Bashaw
FIGURE 8 (1) Superior rectal vein; (2) middle rectal vein; (3) submucus venous
plexus; (4) inferior rectal vein; (5) external rectal sphincter. Source: Ref. 49, p. 119.
number and size of the anastamoses present in each individual’s venous system
the degree of first-pass metabolism in an individual cannot be estimated a
priori. Rectal bioavilability should then be expected to be “intermediate”
that is lying somewhere between that of an intravenous dose and an oral
dose.
No matter if 18th or 21st century technology is used, the primary obstacle
to the delivery of drugs from rectal tissue is that these tissues are not inherently
permeable to drug absorption. The combination of a relatively small surface
area for absorption (~200 cm2) coupled with the small amount of fluid present
and the lack of the specialized structures for absorption (i.e., the villi that
line the small intestine) making the rectal environment a poor one for
absorption to occur.
Because of these factors the primary mechanism for drug absorption in
the rectum, as with the other routes of administration discussed in this chapter
is via passive diffusion. Here, however, drug absorption is dependent not as
heavily on the permeability of the rectal tissue, but on the amount of drug
available in solution ready for absorption. Here the small volume of fluid
present in the rectum and the melting/release of the drug from the suppository
vehicle can play the major role in retarding drug absorption. In some instances,
this can be a desired effect as in the use of controlled-release oral dosage
forms of narcotics placed in the rectum for systemic drug delivery and pain
relief.

Drugs for Rectal Administration

For the most part, the rectal administration of drugs is limited to those drugs
being used to treat a lower GI condition such as constipation, or when the
upper GI tract is compromised either due to disease or for surgical reasons
(i.e., patients awaiting surgery). A review of a standard reference such as the
RED BOOK or the Physicians Desk Reference reveals very few approved
suppository products in the United States. In theory, any drug could be
administered via the rectal route, and in some countries the use of suppository
dosage forms is relatively popular. The relative lack of approved suppository
preparations in the United States is due to a number of factors that are
presented in Table 4.
The primary classes of drugs approved as suppositories in the United States
are the antinauseants (promethazine, prochlorperazine, etc.) and antipyretics
(e.g., acetaminophen). Of the approved agents, acetaminophen, because of
its use in the pediatric population, is the most widely used rectal suppository
in the United States.
TABLE 4 Some PROS and CONS of Rectal Administration

496 Bashaw
Bioavailability and Bioequivalence Considerations
General Design Factors

From a bioavailability/bioequivalence point of view, the precepts to be used
in designing and executing pharmacokinetic trials with suppository dosage
forms are very similar to those surrounding oral dosage forms. Usually rectal
suppositories will represent a line extension of an existing product with which
safety and efficacy has already been demonstrated. In such cases, the
biopharmaceutic program should be concerned not so much with establishing
bioequivalence between the dosage forms, an unlikely occurrence, but in
demonstrating that therapeutic levels can be achieved within a meaningful
therapeutic time window.
As mentioned previously, acetaminophen is the most common rectal
suppository in the United States. In Fig. 9, we see the comparison of an
acetaminophen containing rectal suppository to an oral dosage form, both
dosed at approximately 13mg/kg. As would be expected, the suppository
dosage form produces levels which lag behind and below those produced by
the oral route. Rectal bioavailability was approximately 78% relative to the
oral route, suggesting that a dose of ~16mg/kg would have been required via
the rectal route to provide a similar degree of exposure. These results are
typical of those associated with rectal dosing and reflect the poor nature of
the rectal environment in regard to absorption. It also highlights the fact
that dosing ranges determined from oral dosing may not be relevant with
regard to rectal administration. Independent dose-ranging trials, guided by
the results obtained with oral dosing, should be undertaken to assure that
FIGURE 9 Comparison of an acetaminophen containing rectal suppository to an

oral dosage form, both dosed at approximately 13mg/kg. Source: Ref. 50, p. 427.

when the rectal route of administration is utilized it results in a safe and

efficacious response.
As with oral dosage forms, the in vivo evaluation of suppositories should
include an assessment of dosage form proportionality: Specifically, are the
release characteristics of a drug from different strength suppositories the
same and will these changes have an impact on the clinical utility of the
drug. An example of this type of comparison is contained in Table 5 where
the results of an in vivo biostudy, using acetaminophen suppositories along
with an oral reference dose, are compared for two different strength
suppositories. It is clear from this data that the larger 1000 mg suppository
actually delivers less drug, albeit for a more prolonged period (note the Tmax
difference), than the 2×500 mg suppository treatment. The authors speculate
that these differences could be related to the larger total surface area to unit
volume/dose for the two-suppository treatment relative to the single
suppository treatment. This increased surface area exposes more of the
suppository for melting/dissolving, thus increasing both the rate and
potentially the bioavailability of the drug substance from the suppository
matrix. In either event, an assessment of dosage-form proportionality is
essential for the development of proper clinical dosing recommendation for
a suppository dosage form.
In vitro Methods
The assessment of in vitro release of drug from suppositories has primarily
been limited to the use of melting tests and the use of modified dissolution
apparatuses (specifically modified flow-through cells). Such tests, while
acceptable from a quality control point of view as a release specification, are
insufficient for the assessment of in vivo bioavailability.
Dosing Instructions
Because of its anatomical location subjects should be counseled or the proper
use, i.e., insertion, of suppositories. Subjects should be well hydrated, and
TABLE 5 Pharmacokinetic Results Following Oral and Rectal Administration of

Acetaminophen to 19 Healthy Adults (mean +/- Std. Dev.)
*Relative to oral dosing. Source: Ref. 54.

498 Bashaw
should have had a bowel movement at least an hour prior to insertion to

minimize both the loss of drug to rectal contents (adsorption) and the potential
to trigger a bowel movement by inserting the suppository. The subject should
be instructed to lie on their left side with the left leg straight and the right leg
bent up towards the chest. In adults, the suppository should be inserted 2–3
inches into the rectum with lesser insertion distances being used in children
depending upon their age. After insertion and retention of the suppository,
the subject should be instructed to remain in this position for at least 20min
prior to engaging in other activities.
CONCLUSIONS
As has been shown in this chapter, the development of alternative routes of
drug delivery require careful consideration of the disease state to be treated,
the physiochemistry of the formulation, and the site and manner of drug
application/delivery. Although, physically, widely separated, the intranasal,
topical, and rectal routes of administration share certain similarities in that
the tissues associated with these routes are not normally thought of as sites
of drug absorption. Because of this, drug development for these alternative
routes requires a thorough knowledge of both the disease state being treated
with regard to effective plasma levels and the time course of their attainment.
It is because of the limitations that these routes of administration place on
absorption that one often needs a separate dosing strategy to ensure efficacy
consistent with oral dosing. In vitro methodologies, while useful in lessening
the regulatory burden with the oral route of administration, are less applicable
here due to both methodological short-comings and the lack of a
demonstrated correlation with in vivo events.
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21
Scientific and Regulatory Issues in
Development of Chiral Drugs
Jyoti Chawla
University of Washington
Seattle, Washington, U.S.A
Indra K.Reddy
University of Arkansas for Medical Sciences
Little Rock, Arkansas, U.S.A
BACKGROUND
General principles of drug development are to conduct experiments and

clinical studies which provide the information necessary to assess drug’s
safety, efficacy, and dosage adjustments to make in specific population.
Having chirality in the drug molecule being developed adds additional
challenges which should be resolved. This chapter will provide a brief
introduction to chirality and its implications on pharmacokinetics and
503
pharmacodynamics. Further, regulatory considerations for chiral drugs will

also be discussed.
TERMINOLOGY
Chiral vs. Achiral
Chirality is a geometric attribute; a molecule or object which is not identical
to (or nonsuperimposable upon) its mirror image molecule or object is said
to be chiral. By the same criteria, a molecule or object is said to be achiral if
it is identical to (or superimposable upon) its mirror image molecule or
object. More simple definition for chiral molecule can be stated as “a
molecule that contains one or more asymmetric centers within its molecular
structure” or “molecules that have at least a pair of enantiomers.”
Stereoisomers
Stereoisomers can be defined as molecules consisting of the same chemical
constituents (or groups) with the same structural formulas but differ only
with respect to the spatial arrangement of certain atoms or group of atoms
[1]. They can be subclassified into: (a) optical isomers and (b) geometrical
isomers. Optical isomers are a set of Stereoisomers, at least two of which are
optically active or chiral. Geometric isomers, on the other hand, are
members of set Stereoisomers that contain no optically active centers.
Enantiomers
Two Stereoisomers in which molecules are nonsuperimposable mirror
images of one another are said to be enantiomers. Enantiomers differ only in
the spatial arrangement of ligands attached to the chiral center, but they
share the same physicochemical properties such as refractive indices,
melting points, boiling points, and solubility. Enantiomers are sometimes
referred to as optical antipodes, where anti means opposite and podes
means feet.
Diastereoisomers
Stereoisomers with two or more asymmetric centers and whose molecules
are not mirror images of one another are said to be diastereoisomers, or
simply diastereomers. Unlike enantiomers, diastereomers can differ in
physicochemical properties such as signs and magnitudes of optical
rotations, melting points, solubilities, and refractive indices. The most
common diastereomeric molecule is one that contains two asymmetric

Scientific and Regulatory Issues 505
carbons. This situation is illustrated by the compounds ephedrine and

pseudoephedrine. Each diastereomer of ephedrine and pseudoephedrine
exists as a member of an enantiomeric pair, i.e., d- and 1-ephedrine and d-
and 1-pseudoephedrine, respectively. Thus, diastereomeric molecules with
two asymmetric centers are most often represented by four stereoisomers.
Diastereoisomers and geometric isomers are both chemically distinct and
pharmacologically different. They are generally readily separated without
chiral techniques.
Racemic Mixture
An equal (1:1) mixture of two enantiomers is said to be racemic. The IUPAC
rules [2, 3] state that “when equal amounts of enantiomeric molecules are
present together, the product is termed racemic independently of whether it
is crystalline, liquid or gaseous.” Thus in the IUPAC rules the word
“racemic” (adjective) is applied to an optically inactive product in any state
of matter, and “racemic mixture” would appear to be the correct
terminology for a 1:1 mixture of enantiomers in any physical state. A
racemic mixture, therefore, is a 50:50 mixture of the two enantiomers of a
chiral compound. Conversion of one enantiomer to a 1:1 mixture of the two
is referred to as racemization. Because the two enantiomers have equal and
opposite specific rotations, a racemic mixture has a specific rotation of zero,
i.e., it is optically inactive. In nature, most naturally occurring compounds
occur as a single enantiomer, not as racemic mixtures. The importance of
racemic mixtures is that ordinary laboratory synthesis which generates a
stereogenic center produces a racemic mixture.
Optical Activity
A physical property that distinguishes two enantiomers is “optical activity,”
which refers to the property of chiral compounds of rotating the plane of
plane-polarized light to the right (clockwise) or to the left (counter-
clockwise). The two enantiomers have exactly the same ability to rotate the
plane of monochromatic plane-polarized light, quantitatively, but they
rotate it in opposite directions. Thus, if one enantiomer rotates the plane by
10 degrees clockwise (considered a positive rotation), the other rotates it by
-10 degrees in the counterclockwise direction (considered a negative
rotation). Since the exact amount of the rotation of the plane by a given
enantiomer depends upon how much of that enantiomer the light
encounters as it passes through the solution, the measured rotation is
divided by the concentration of the enantiomer and by the path length of the
polarimeter cell to give a true measure of the inherent ability of the
enantiomer to rotate the plane of polarized light. A positive rotation is also

referred to as dextrorotation and a negative rotation is called levorotation,

and denoted by d and 1 respectively, and the terms dextrorotatory and
levorotatory are superseded by (+) and (-), respectively [1].
NOMENCLATURE OF STEREOISOMERS
The Fischer Convention [4]
The configuration of an asymmetric center was initially determined by the

chemical transformation of the chiral molecule to an arbitrarily selected
standard, (+)-glyceraldehyde. This was the basis of the Fischer Convention
for the determination and designation of configuration. The system operates
by relating the configuration at the asymmetric center of the molecule under
investigation to (+)-glyceraldehyde, which was arbitrarily assigned the D
configuration. To assign a configuration, the molecule under investigation
must be chemically converted to glyceraldehyde or to another molecule of
known configuration. After this is accomplished, the sign of rotation is
determined and the D or L configuration is assigned accordingly. The sign of
rotation cannot be employed prior to assigning a configuration, because
they do not always correspond. For example, L-alanine has a (+) sign of
rotation, whereas the sign of rotation for L-glyceraldehyde is (-). The Fischer
Convention is widely used to assign a configuration for sugars, which
contain a number of asymmetric centers. For diastereomers with only two
centers, the Fischer Convention assigns a series as D or L according to
whether the configuration at the highest numbered asymmetric center is
analogous to D- or L-glyceraldehyde. The Fischer Convention is often
incorrect and difficult to use, especially when complex chemical transforma-
tions are required to convert the molecule under investigation into a
molecule of known configuration. In addition, the assigned configuration,
D or L is often confused with the observed sign of rotation, d or 1. Because
of the potential confusion that it could lead, the Fischer Convention has
been almost entirely replaced by the Cahn-Ingold-Prelog Convention.
The Cahn-Ingold-Prelog Convention [5]

The Cahn-Ingold-Prelog Convention was designated by its originators as
the “sequence rule,” since it designates the sequence of substituents around
the asymmetric center. In this method, the substituents at the chiral center
are first sized according to their atomic number from the largest to the
smallest. Once the rank order is determined, the molecule is held so that the
lowest group in the sequence is pointed away from the observer. Then if the
other groups listed in the descending order of precedence are oriented

clockwise, the molecule is designated R (rectus), and if counterclockwise, S

(sinister). In the example presented in Fig. 1, the order is L (large), M
(medium), S (small), and S’ (smallest). The molecule is then oriented so that
the smallest (S’) substituent is directed away from the observer. The
configuration is then determined by whether the sequence L-M-S goes in a
clockwise or counterclockwise direction. A clockwise direction is designated
as R (rectus) whereas the counterclockwise direction is designated as S
(sinister). This convention can be used to rapidly and unambiguously
specify the configuration of a chiral center. If one enantiomer has an R
designation, its antipode or mirror image has the S configuration. The
Cahn-IngoldPrelog Convention (see Fig. 1) is also very useful for describing
diastereomers. In the case of diastereomers, each chiral center is designated
independently and the configuration of the whole molecule can be
conveniently assigned. For example, instead of d- and 1-pseudoephedrine,
the assigned configurations are (R, S)- and (S, R) ephedrine and (R, R)- and
(S,S)-pseudoephedrine. The enantiomeric relationships within the ephedrine
and pseudoephedrine molecules and the diastereomeric relationship
between ephedrine and pseudoephedrine are recognized clearly.
A set of terms are also in use to describe the pharmacological activity of
stereoisomers. In an enantiomeric pair, the isomer with the greater
pharmacological affinity or activity is known as eutomer, and the one with
the lower pharmacological affinity or activity is called distomer [6]. The
ratio of affinities or activities of eutomer to distomer is referred to as the
eudismic ratio, and the logarithm of eudismic ratio is known as eudismic
index. Slope of a plot of eudismic index vs. the logarithm of affinity of
eutomer (ideally expressed either pA2 or pD2 values in pharmacology, or Ki
or Km values in enzymology) for a homologous series is called the Eudismic
Affinity Quotient (EAQ). It represents a quantitative measure of the
stereoselectivity within compound series for a specific biological effect [7,
8]. The greater the difference in pharmacological activity between a pair of
enantiomers, the greater will be the specificity exhibited by eutomer, and
this is referred to as Pfeiffer’s Rule. A positive slope of EAQ reflects such
FIGURE 1 Cahn-lngold-Prelog Convention.

greater difference. However, it should be noted that exceptions to Pfeiffer rule

have been reported [9].
STEREOSELECTIVITY
Stereoselectivity (or enantioselectivity) in pharmacology as well as
pharmacokinetics following administration of racemic drug has been
recognized since early part of last century. In the past few decades,
pharmacological and pharmacokinetic investigations have clearly demon-
strated significant differences in the biological activity of some isomeric
pairs. Following is a concise review of Stereoselectivity with regard to
pharmacodynamics and pharmacokinetics of racemic drugs.
Pharmacodynamic Considerations
From the pharmacodynamic and therapeutic standpoint, multiple outcomes
are possible with racemic drugs. Following is a brief discussion on three
categories of racemic drugs based on the qualitative and quantitative
activities of stereoisomers. It should be noted that many drugs may belong
to more than one category, and with ever-growing knowledge of
stereochemistry of drug action and disposition, they may be more
appropriately placed into the relevant category.
Racemates in which one Stereoisomer Possesses the Majority or all of

the Beneficial Activities and the Other Isomer is Inactive
It is less common, although highly desirable, to have all the activity in one
enantiomer. This necessitates development of single isomer, avoiding the
unwanted activity/toxicity of the antipode. Selected examples where one
member of an enantiomer pair was pharmacologically active and the other
inactive include α-methyldopa (antihypertensive activity) [10, 11] and
propranolol (β-blocking activity) [12]. In the case of beta-blockers
representing the aryloxyproponolamine category, the therapeutic effect
resides almost entirely in the S-stereoisomer. For example, the eudismic ratios
of three beta-blockers, atenolol, propranolol, and metoprolol, are 12, 130,
and 270, respectively. The inactive (or less active) enantiomers of these beta-
blockers are not known to cause any serious side effects.
Racemates in which both enantiomers have similar potency
Although it is quite common for enantiomers to possess similar qualitative
pharmacological activity, it is uncommon that both isomers possess similar
qualitative and quantitative activity profiles. Examples where similar
qualitative activity was observed for many enantiomeric pairs, some of
which include promethazine (with respect to antihistaminic activity) [13],

flecainide (with respect to electrophysiological effects) [14], warfarin

(anticoagulant activity) [15, 16], and verapamil (vasodilator effects) [17,
18]. In such cases with racemic drugs, the separation of two enantiomers
may not be justified. However, although the two enantiomers may be
qualitatively and quantitatively similar with respect to the main therapeutic
activities for which they are indicated, subtle differences with regard to
other activities are possible which must be carefully addressed.
Both enantiomers qualitatively and quantitatively differ in their activity
Stereoisomers may sometimes exhibit desirable, but different biological
effects such that both may be marketed with different therapeutic
indications. For example (8S, 9R), quinine is an effective antimicrobial
agent, while the corresponding (8R, 9S) diastereomer quinidine is an
antiarrhythmic agent. Other examples of enantiomers that have completely
different (qualitative) activities include propoxyphene (the d-isomer has
analgesic activity and the 1-isomer exhibits the antitussive properties) and
sotalol (where the d-isomer is a type 3 antiarrhythmic while 1-sotalol is a ß-
blocker). The two optically active isomers of indacrione have qualitatively
and quantitatively different diuretic and uricosuric activities [19].
Sometimes Stereoisomers possessing different pharmacodynamic activities
may be developed as racemates because the combination offers a
therapeutic advantage. For example, (R)-enantiomer of indacrinone has a
diuretic activity and causes uric acid retention, whereas the S-enantiomer
possesses uricosuric activity and promotes the secretion of uric acid. This
combination may be beneficial to induce diuresis in hypertensive patients
who typically have elevated uric acid levels.
Pharmacokinetic Considerations
Absorption
Drugs, in general, are absorbed by passive diffusion, a process dependent
upon physicochemical properties of diffusant molecule such as aqueous/
lipid solubility, ionization, and molecular size. Since enantiomers do not
exhibit differences in their physicochemical properties, stereoselectivity is
not expected. However, diastereoisomers may exhibit differences in their
absorption profies as they differ in their physicochemical properties. Drugs
that are transported via carrier-mediated mechanisms (e.g., facilitated
diffusion or active transport processes) may exhibit significant stereoselec-
tivity. This is because the process of carrier-mediated transport involves a
specific interaction of the drug with a chiral endogenous macromolecule.
For example, it has been reported that L-isomer is preferentially absorbed
compared to the D-enantiomers for dopa and methotrexate [20, 21]. The
transport systems involving P-glycoprotein-mediated efflux mechanisms are

also potentially stereoselective. Interestingly, some stereoisomers have been

shown to facilitate the absorption of their optical antipodes. For example,
the bioavailability of S-propranolol is greater when administered as a
racemate than as a single isomer, suggesting that R-propranolol promotes the
absorption of S-isomer [22].
Distribution
As with drug absorption, distribution of drugs is generally described by
passive diffusion. Stereoselectivity in drug distribution may occur as a result
of binding of drugs to either plasma or tissue proteins and/or transport via
specific tissue uptake and storage mechanisms. Difference between
enantiomers in plasma protein binding have been reported for a number of
drugs. A majority of drugs bind in a reversible manner to plasma proteins,
notably to human serum albumin (HSA) and/or alpha1-acid glycoprotein
(AGP). Acidic drugs bind preferentially to HSA, with binding at site II
(benzodiazepine site) on the protein generally displaying greater
enantiomeric differences than at site I (warfarin site) and basic drugs
predominately bind to AGP. It should be noted that Stereoselectivity in
binding may vary for different proteins, e.g., the protein binding of
propranolol to AGP is stereoselective for the S-enantiomer, whereas binding
to HSA favors (R)-propranolol [23]. In whole plasma the binding to AGP is
dominant such that the free fraction of the R-enantiomer is greater than that
of (S)-propranolol.
Enantioselective tissue uptake, which is in part a consequence of
enantioselective plasma protein binding, has been reported. For example,
the uptake of ibuprofen into lipids is stereoselective in favor of the R-
enantiomer, but this is as a result of stereospecific formation of the acyl-CoA
thioester followed by incorporation as hybrid triglycerides [24].
Metabolism
Drug metabolism, involving phase I as well as phase II biotransformations,
shows Stereoselectivity. Enantioselectivity in drug metabolism may be
described as the rule rather than the exception and probably is responsible
for the majority of the differences observed in enantioselective drug
disposition. Stereoselectivity in metabolism may arise due to differences in
the binding of enantiomeric substrates to the enzyme active site and/or be
associated with catalysis due to differential reactivity and orientation of the
target groups to the catalytic site. As a result, a pair of enantiomers are
frequently metabolized at different rates and/or via different routes to yield
alternative products. Examples include propranolol, verapamil, and war-
farin. For example, S-isomer of propranolol is metabolized predominantly

by glucoronidation, whereas R-isomer undergoes oxidative degradation to

form 4-hydroxypropranolol. Enantioselectivity in metabolic clearance is
more apparent for drug molecules undergoing first-pass enterohepatic
metabolism.
The stereoselectivity of drug metabolic processes may be classified into
three categories in terms of their selectivity with respect to the substrate, the
product or both. An alternative classification involves the stereochemical
consequences of the transformation reaction, and according to this
approach, metabolic pathways may be divided into five groups: (a)
prochiral to chiral transformations, (b) chiral to chiral transformations, (c)
chiral to diastereoisomer transformations, (d) chiral to achiral
transformations, and (e) chiral inversion.
Chiral Inversion. The process of metabolic conversion of one stereoisomer

into its enantiomer with no other alteration in structure is known as chiral
inversion. Examples of agents undergoing this type of transformation are
the 2-arylpropionic acid (2-APAs) nonsteroidal antiinflammatory drugs
(NSAIDs) such as ibuprofen, fenoprofen, flurbiprofen, ketoprofen [25] and
the related 2-aryloxypropionic acid herbicides, e.g., haloxyfop [26]. In the
case of the 2-APAs the reaction is essentially stereospecific with the less
active, or inactive, R-enantiomers undergoing inversion to the active S-
enantiomers. Following administration of (S)-stiripentol the R-enantiomer
produced by racemization undergoes conjugation with glucuronic acid and
excretion in the bile, the S-enantiomer appearing in the systemic circulation,
whereas following administration of (R)-stiripentol the glucuronidation
pathway is saturated and both enantiomers, (S)-stiripentol being formed in
the gastric acid, are found in the systemic circulation [27]. For drugs
exhibiting chiral inversion, the residence time of the drug in the
gastrointestinal tract affects the eudismic ratio. As an example, the relative
concentration of the pharmacologically active S-enatiomer of ibuprofen (S:R
ratio) increases with prolongation of the GI transit time of racemic
formulations due to a corresponding increase in chiral inversion of the R- to
S-enantiomer in the gut. In such cases, administration of S-ibuprofen and not
the racemate, therefore, reduces the formulation-dependant variability in the
concentration of the active enantiomer in the body.
Renal Clearance
Stereoselectivity in renal excretion may occur with all aspects of renal
clearance including protein binding, glomerular filtration and passive
reabsorption, or active secretion or reabsorption. Enantioselectivity in renal
clearance has been reported for a number of drugs and in many cases the
selectivity is relatively modest with enantiomeric ratios between 1.0 and

3.0. The diastereoisomers quinine and quinidine show enantioselectivity in

renal clearance where the difference is about four fold with values of 24.7
and 99 mL min-1 in man, respectively [28]. In another case, concurrent
administration of probenecid has been shown to stereoselectively reduce
the renal clearance of (-)-isomer of sultopride, but not that of the (+)-
enantiomer following administration of the racemic drug to rats [29]. In
contrast, coadministration of the racemic drug with procainamide lead to
significant reductions in both total and renal clearance of both the
enantiomers [29]. Stereoselective renal clearance may also occur for
metabolites. For example following the repeated oral administration of the
individual enantiomers of disopyramide, significant differences in both the
total and unbound renal clearances of the monodesisopropyl metabolite
were observed, both processes being Stereoselective for the (+)-S-enantiomer
[30]. In contrast the total renal clearance for the drug showed no
stereoselectivity, whereas the unbound renal clearance of (S)-disopyramide
was greater than that of the R-enantiomer. The renal elimination of both
enantiomers of both the compounds was associated with tubular secretion
and the possibility exists that drug-metabolite-enantiomer interactions in
renal tubular secretion may occur [30]. Stereoselective elimination may
greatly influence pharmacodynamic parameters, including intensity and
duration of action for drugs eliminated primarily by renal clearance. From a
clinical standpoint, a less potent, but slowly cleared isomer offers greater
advantage than a highly potent, rapidly cleared enantiomer.
Protein Binding
Enantiomers of many chiral drugs have shown differential affinities toward
human plasma proteins. Much of the drug, in general, bind to different
extents to one or more of the different blood elements such as cells and
proteins when reach the systemic circulation. Protein binding of some
enantiomers to plasma proteins, albumin, and alpha1-acid glycoprotein
(AAG) may be Stereoselective. The high affinity binding sites on albumin
have more receptor-like properties than the binding sites on α1-acid
glycoprotein, since the former can more effectively differentiate between
different drug enantiomers than the latter. Acidic drugs, such as warfarin
and active metabolites of diazepam and oxazepam, bind stereoselectively to
serum albumin, whereas basic drugs such as verapamil and disopyramide
bind stereoselectively to AAG [31–33]. For drugs exhibiting enantioselective
protein binding, one should carefully evaluate the dynamics of the racemic
mixture to determine the concentration of the free, unbound drug at the
target site to assess its clinical activity and toxicity.

REGULATORY CONSIDERATIONS
Despite the challenges identified with some racemates, the common practice
of developing drug products of racemates has led to an ongoing discussion
on the rationale and the regulatory aspects of chiral drug product
development by the scientific community [34–39]. This section presents a
discussion on regulatory issues relating to the pharmaceutical development
of stereoisomers, particularly those with one or more chiral centers.
Guidelines for development of chiral drugs have been issued by European,
Candian, United States, and other regulatory agencies [40–42]. Some of the
guidance documents are (1) FDA’s policy statement for the development of
new stereoisomeric drugs, issued by the FDA in 1992. (2) Bioavailability
and Bioequivalence Studies for Orally Administered Drug Products—
General Considerations (March 2003). (3) Investigations of chiral active
substances issued by commission of the European countries in 1994. (4)
Stereochemical issues in chiral drug development, issued by Therapeutic
Product Programme, Canada (2000).
As discussed earlier, stereoisomers are often readily distinguished by
biological systems and may exhibit different pharmacokinetic properties
including absorption, distribution, metabolism, and excretion. Conse-
quently, quantitative and/or qualitative differences in pharmacologic and/
or toxicologic effects are possible with racemic drugs. When stereoisomers
are biologically distinguishable, they may behave as different drugs.
Regardless of this behavior, it has been past practice to develop chiral drug
products as racemates. There are many reasons for such a practice. Some of
the products that are racemates were marketed at a time when good
separation and/or synthetic procedures for individual enantiomers were not
available for manufacture on a commercial scale. Some of these products
date to before 1938 when extensive new drug applications (NDAs) were not
required for marketing of a new drug. In some cases, enantiomers were
found to be identical in pharmacological properties. In other cases, one
enantiomer was inert or possessed little or no biological activity. Since
commercial separation of racemates was less common, the question of
developing individual enantiomers as drug products was largely of academic
interest. The technological advances over the past 25 years, including
largescale chiral separation procedures or asymmetric synthesis, make it
possible to produce many single enantiomers on a commercial scale.
Consequently, the need for the regulatory policies and guidelines with
respect to the development of stereoisomeric mixtures has grown over the
years. It follows that the development of chiral drugs presents a number of
issues, each of which is recognized as an important consideration [40].
These may include:

• acceptable manufacturing control of synthesis and impurities

• acceptable enantiomeric assays
• adequate pharmacologic and toxicologic assessment
• proper characterization of metabolism and distribution
• assessment of chiral inversion
• appropriate clinical evaluation
Among the stereoisomers, geometric isomers and diastereomers should be

treated as separate drugs and developed accordingly. However, with the rare
exception of cases where in vivo interconversion occurs, the development of
mixtures of geometric isomers or diastereomers is generally not justified
unless they, by chance, represent a reasonable fixed dose combination [41].
In such cases, whether the optimal ratio of the two isomers is the ratio
produced by an unmodified synthesis should be carefully examined.
Geometric isomers, in general, have been developed as single isomers,
whereas practice with respect to diastereomers has been variable.
Since most biochemical processes are stereospecific, chiral substances
from natural sources are normally obtained in an optically active form. For
example, antibiotic products prepared by fermentation are mostly
stereospecific. Products prepared by different biochemical processes,
however, may have different configurations. Lactic acid produced by
fermentation of sugars is levorotatory, while lactic acid produced in living
muscle is dextrorotatory [42].
All of the reported synthetic procedures of steroids at one time yielded
racemic mixtures. Asymmetric processes were developed for many single
enantiomers, some of which employed yeast. Some pharmaceutical firms
used both microbiological fermentation and chemical transformations to
produce the specific enantiomer. Both of these forms are still on the market
[42]. A completely synthesized product, the antihypertensive drug
methyldopa is prepared completely as the levo form, since all of the activity
lies in it and not with the other enantiomer.
Racemates vs. Enantiomers

Pharmacological assessment of chiral substances in an early research phase
can facilitate the selection of either single enantiomer or racemate for drug
product development. The pharmacological investigations of enantiomers
may reveal different scenarios, some of which are discussed earlier. While
inactivity of one member of an enantiomeric pair might be considered trivial
and often overlooked, there are instances in which toxicity has been
associated with one member of a pair of stereoisomers, not necessarily the
active isomer (eutomer). For example, vomiting is caused by the d-isomer of
levamisole and myasthenia gravis symptoms have disappeared when the d-

isomer was removed from d, 1-carnitine. In case of ketamine, the S(+)-

isomer is three to four times more potent than R(-)-enantiomer with respect
to the desirable anesthetic and analgesic activities, but the notorious side
effects were overwhelmingly linked to R(-)-isomer. Further, there are many
cases in which enantiomers have exhibited pharmacokinetic differ-ences, the
discussion of which is beyond the scope of this chapter. While
pharmacological studies have presented us with many different situations
for racemates, it is incorrect to expect that the concentration of enantiomers
in plasma remain 1:1. Further, it is unreasonable to assume that the
optimum ratio of the enantiomeric pair to be the 1:1 ratio of the racemate.
General Policy and Application Submissions for Chiral

Drugs
When stereoisomers are considered for drug product development, the
sponsor must first decide as to whether to separate the isomers (or
synthesize them individually) or to deal with the substance as a racemate for
all drug development investigations. These decisions must take into
consideration the number of isomers present, the difficulty of separation (or
synthesis as the case may be), and the toxicity/effectiveness of the substance.
Other considerations in the selection of a particular form of stereoisomers
include the route of administration, rates of absorption, mechanism of
action, biotransformation, elimination, and biological activity of the
isomers.
The stereoisomeric composition of a drug with a chiral center and the
quantitative isomeric composition of the material used in pharmacologic,
toxicologic, and clinical studies should be known. The final product
specifications should assure identity, strength, quality, and purity from a
stereochemical point of view.
In order to evaluate the pharmacokinetics (i.e., kinetics of absorption,
distribution, metabolism, and excretion) of a single enantiomer or mixture
of enantiomers, it is important that one should develop quantitative assays
for individual enantiomers in in vivo samples early in drug development.
Any potential interconversions between the enantiomers should be carefully
evaluated. Failure to take interconversion into account while developing a
single enantiomer can result in drug development failure. For example, a
racemate which was approved and had efficacy residing in one isomer was
being developed as an enantiomer. The sponsor initiated developing active
enantiomer using 50% of the dose that was approved as an racemate.
However, the fact that, in vivo, about 20% of inactive enantiomer converts
to active enantiomer was ignored. Thus, while developing active isomer,
60% of the racemate dose should have been used (to compensate for the
interconversion). Since interconversion was not taken into account, it

resulted in an inconclusive trial. Thus, it is important to take interconver-

sion into account. In general, when the pharmacokinetic parameters of
isomers of the racemate drug product are different, manufacturers should
monitor the enantiomers individually to determine properties such as dose
linearity, the effects of altered metabolic or excretory function, and
drugdrug interactions. If the pharmacokinetic profiles for both
stereoisomers are found to be identical or a fixed ratio between the plasma
levels of enantiomers, an achiral assay or an assay that monitors one of the
isomers should be adequate for subsequent evaluation.
If and when possible, the main pharmacologic activities of the
stereoisomers should be compared in in vitro systems, in animals, and/or in
humans. A relatively mild toxicologic profile of a chiral chemical using the
racemate would, in general, support further development without separate
toxicologic evaluation of the individual enantiomers. However, if there is
any toxicity beyond the natural extensions of the pharmacologic effects of
the drug, toxicologic evaluation of the individual enantiomers should be
undertaken [40].
While the decision of whether to market a specific isomer or a racemate is
one that is primarily under the control of the pharmaceutical firm (or
sponsor), it is generally based on the pharmacologic, therapeutic, and
toxicological considerations of the intact racemate, individual isomers,
stability of the drug, technical feasibility of manufacturing the individual
isomer on a commercial scale, and cost of manufacture of individual
isomers. Enantioselectivity in pharmacokinetics and/or pharmacodynamics
presents four possible combinations of scenarios, that is, neither PK nor PD
are enantioselective; only PK or PD are enantiospecific; or PK and PD are
enantioselective. When PD (safety and efficacy) of a racemate is
enantioselective, one needs to consider if developing an enantiomer is a
better option.
When a sponsor submits an IND for either the racemate or the individual
isomer, it would be very helpful to the reviewers in the regulatory agencies to
have a discussion on why a particular form was chosen to be included in the
submission. In some cases, studies of individual isomers have been
undertaken as an after-the-fact decision when clinical findings have shown a
serious adverse reaction together with an effective response in a particular
disease or condition. Early testing of the individual isomers on their
pharmacological and toxicological properties would provide informa-tion,
which would help the sponsor make a decision on how to proceed with the
product development. Should the decision be to develop the racemate,
adequate controls and tests must be used to assure that the drug used in
animal testing and human trials is identical to that proposed for marketing,
and that it can be reproduced in every batch manufactured. Subsequent to
the IND submission, FDA invites discussion with sponsors concerning

TABLE 1 Required Information for Chiral Drug Submissions: Chemistry,

Manufacturing, and Controls
Source: Ref. 40.

whether to pursue development of the racemate or the individual

enantiomer and general drug development plan.
The information that is presented in the IND submission must detail the
full composition of the drug substance, and include adequate information
on the method of manufacture, the starting materials, intermediates,
reagents, solvents, catalysts, in-process controls, and final controls. It is
imperative that the information on whether the drug is a specific
enantiomer, a racemate, or a mixture should be provided. The data
submitted on substances that exist as stereoisomers should include a
discussion on the possible isomers that may result from the method of
manufacture, and the results of studies carried out to investigate the
physical, chemical, and biological properties of these isomers. Since
enantiomeric differences are common between the different animal species
and between animals and humans, as evidenced by the permeation of
propranolol enantiomers, it should- be clearly mentioned as to what form
was used in the animal studies and what form(s) will be used in the initial
use in humans [40]. As stated earlier, drugs in which one of the isomers in a
racemate is “inactive” with respect to safety and adverse events, an isomer
may be developed for marketing, provided the separation or asymmetric
synthesis is economically prohibitive or technically difficult on the large
scale. The information that should be generally provided by the sponsor in
the drug application submissions, in part, is presented in Table 1.
Development of a Single Stereoisomer After Studies on Racemate

When developing a single Stereoisomer from a racemic mixture that has
already been studied nonclinically, appropriate pharmacologic/toxicologic
evaluation should be carried out to permit the existing information
generated on the racemate to be applied to the pure enantiomer.
Continuation of investigations usually include the repeat-dose toxicity
evaluation carried out up to three months and the reproductive toxicity in
the most sensitive species, using the single enantiomer. A positive control
group consisting of the racemate should be included in these studies. If the
toxicological profiles of the single enantiomer product and the racemate are
similar, no further studies would generally be required. However, if the
single enantiomer is found to be more toxic, further investigation should be
conducted to offer explanation for that finding and the implications for
human dosing should be considered [40].
If the pharmacodynamic and pharmacokinetic differences between the
enantiomers are insignificant, racemates may be considered for develop-
ment. However, development of a single enantiomer may be desirable in
some cases where, for example, significant differences in toxic or
undesirable pharmacologic effects are seen. The pharmacological and

toxicological profiles of the individual enantiomers and their active

metabolites should be further investigated if the toxicity observed with the
racemate at clinical doses is not anticipated from the pharmacology of the
drug.
It is important that both the enantiomers should be evaluated clinically
and based on these findings, one should consider a racemate or individual
enantiomer. When both the enantiomers are pharmacologically active but
differ significantly in potency, specificity, or maximum effect, only one
isomer should be considered for development. When both the enantiomers
exhibit desirable but different properties, development of a mixture of the
two, not necessarily the racemate (componds with 1:1 ratio of enantiomers),
as a fixed combination might be reasonable [40].
If a racemate is considered for development, the pharmacokinetics of the
two enantiomers should be investigated in Phase 1 studies. Any potential
interconversion should also be studied. Based on Phase 1 or 2
pharmacokinetic data, it would be possible to determine whether an achiral
assay or monitoring of just one enantiomer where a fixed ratio is confirmed
will be sufficient for pharmacokinetic evaluation. If a racemate has been
marketed and the sponsor desires to develop the single enantiomer,
evaluation should include determination of whether there is significant
conversion to the other isomer, and whether the pharmacokinetics of the
single isomer are the same as they were for that isomer as part of the
racemate [40].
Use of Enantiospecific Assays for Assessing Bioavailabilty and

Bioeqivalence
Use of enantiospecific assays to assess bioavailabilty and bioequivalence has

received considerable attention in the literature. Guidance published by the
FDA “Bioavailability and Bioequivalence Studies for Orally Administered
Drug Products—General Considerations” addresses this issue and is
summarized in Fig. 2. Regulatory guidances on chiral drug development
issued by other countries have also addressed the issue of using
enantiospecific assay.
In general, for bioavailability studies, measurement of individual
enantiomers may be important. For bioequivalence studies, the FDA
guidance recommends measurement of the racemate using an achiral assay.
Measurement of the individual enantiomers in bioequivalence studies is
recommended only when all of the following conditions are met (Fig. 2): (1)
the enantiomers exhibit different pharmacodynamic characteristics; (2) the
enantiomers exhibit different pharmacokinetic characteristics; (3) primary
efficacy/safety activity resides with the minor enantiomer; and (4) nonlinear

FIGURE 2 Decision tree for use of stereospecific assay for BE studies.
absorption is present (as expressed by a change in the enantiomer

concentration ratio with change in the input rate of the drug) for at least one
of the enantiomers.
Guidance issued by Therapeutic Products Program (Canada) states that
in general, when comparing solid dosage forms of similar type (e.g., two
immediate release formulations), total drug concentrations can be
measured. Bioequivalence comparisons should be made between
“pharmaceutically equivalent products.” The bioavailability of each
enantiomer should be compared in the following cases:
a. bioequivalence studies for comparison of different types of solid
oral dosage forms, e.g., comparison of a modified release drug
product to an immediate-release product, or to a different kind
of modified-release formulation.
b. If the in vivo enantiomeric ratio is affected due to differences in
release rates or absorption of the drug substance, or if the drug
shows enantioselective nonlinear first-pass metabolism.

CLINICAL PHARMACOLOGY AND BIOPHARMACEUTICS:

CASE STUDIES
Presently, there are several racemates and individual enantiomers of

previously approved racemates being marketed. Some of the examples
include citalopram, which is a racemate, escitaloprarn (the S-isomer of
citalopram), esomeprazole (S-isomer of omeprazole), and focalin (the
dextrorotary isomer of methylphenidate), which are the single isomers of
already-approved racemates, etc.
Due to space limitations in this book, we cannot get into details of what
kind of studies were submitted for approval of these products. However,
readers can learn a great deal about the regulatory submission for any drug
by refering to the drug product label and reviews posted on the FDA website
for these drugs.
EXCLUSIVITY PERIOD FOR ENANTIOMER OF PREVIOUSLY

APPROVED RACEMATES
It is not required to demonstrate the contribution of each isomer to the
effectiveness of the racemic drug being proposed for marketing. Therefore
combination drug policy as described in 21 CFR 300.50 is not applied to
chiral products. Since combining of the two enantiomers in a racemate drug
product is not deliberate, the activities of the enantiomers are usually
similar, and in the past the separation was difficult, therefore the
combination drug policy is not applied to racemic drugs. However, the
mixtures of diastereoisomers are readily separated, and their activities are
often very different and therefore are considered as combination drugs and
subject to the combination drug policy. At present, marketing exclusivity
period for developing a single isomer of previously approved racemate is
three years. FDA requested comments (62 FR 2167, January 15, 1997) on
the appropriate period of marketing exclusivity for drug products whose
active ingredient is a single enantiomer of a racemate that is an active
ingredient of a previously approved drug product. Several varied responses
were received by the FDA and have been summarized elsewhere [43].
SUMMARY
Stereoisomers is a general term used for molecules that are identical in

atomic constitution and bonding, but differ in the orientation of the atoms
in space. Literature shows numerous examples of drugs where enantiomers
of a racemate show differences in pharmacology, pharmacodynamics,
pharmacokinetics, metabolism, toxicity, protein binding, etc. With some

drugs, one enantiomer may show an entirely different pharmacological

response, or may be inactive or less active than the other enantiomer. There
may be differences in the degree of toxicity, or different toxic responses may
be produced by the pair of enantiomers. When pharmacodynamics and/or
pharmacokinetics differences exist between isomers, it can create a
significant challenge in interpretation of the activity, if achiral blood level
assays are used. Advances in chiral chemistry (manufacturing and
analytical) technique have led to a possibility of producing single
enantiomer on a commercial scale, and measuring individual isomer levels
in biological fluids. The drugs which show enantioselective PK and/or PD
add a challenge to the known principles of drug development. For chiral
drugs, additional considerations are presented in Table 1 and Fig. 2. In
general, for PK assessment of chiral drugs, the main difference (as compared
to drug without a chiral center) is the decision whether to use an
enantioselective or an achiral assay to characterize the pharmacokinetics.
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17. Echizen, H.; Brecht, T.; Niedergesass, S.; Vogelgesang, B.; Eichelbaum, M. The
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Intestine. Nature 1973, 242, 463–465.
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Folate Transporter in Rabbit Small Intestine: Studies with Amethopterin
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22. Lindner, W.R.; Lindner, W.; Rath, M.; Stoschitzky, K.; Semmelrock, H.J.
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26. Bartels, M.J.; Smith, F.A. Stereochemical Inversion of Haloxyfop in the Fischer
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Mechanistic Studies. Drug Metab. Dispos. 1994, 22, 544–553.
28. Notterman, D.A.; Drayer, D.E.; Metakis, L.; Reidenberg, M.M. Stereoselective

Renal Tubular Secretion of Quinidine and Quinine. Clin. Pharmacol. Ther.

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Inhibitory Effects of Procainamide and Probenecid on Renal Excretion of
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Pharmacokinetics of Disopyramide Enantiomers in Humans. Drug Metab.
Dispos. 1988, 16, 858–864.
31. Muller, W.E. Drug Stereochemistry-Analytical Methods and Pharmacology,
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32. Drayer, D.E. Pharmacokinetic Differences Between Drug Enantiomers in Drug
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42. Web site addresses for regulatory agencies:
http://www.hc-sc.gc.ca/hpb/ Canada
http://www.ifpma.org/ichl.html ICH
http://www.mhw.go.jp/english/index.html Japan
http://www.health.gov.au/tga/ Australia
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43. Chandra Sahajwalla. Regulatory Considerations in Drug Development of
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K.Reddy; Reza Mehvar, Eds.; Marcel Dekker: New York, 2003; in press.

22
A Regulatory View of Liposomal Drug
Product Characterization
Kofi A.Kumi and Brian P.Booth

INTRODUCTION
Liposomal drug products are defined as drug products containing drug
substances (active pharmaceutical ingredients) encapsulated in liposomes
[1]. A liposome is a microvesicle composed of a bilayer of lipid amphipathic
molecules enclosing an aqueous compartment [1]. Liposome drug products
are formed when a liposome is used to encapsulate a drug substance within
a lipid bilayer of lipid amphipathic molecules enclosing an aqueous
compartment [1]. Liposomal drug products are a relatively new “class” of
drugs. Doxil (liposomal doxorubicin), for example, was only approved in
late 1995 and there are only a handful of approved products (Ambisome,
Abelcet, Amphotec, Daunosome, Depocyt, Doxil), and a limited number of
newer products are at various stages of development. As a result, regulatory
thinking on these types of products is not as well evolved as it is for more
traditional oral or intravenous formulations. However, the Guidances for
Industry for orally administered products, and the concepts that underlay
them, are also useful guides for our approach to evaluating liposomal
525
526 Kumi and Booth
products [2–4]. Although these agents are generally administered

intravenously, they also share many characteristics with peroral drugs, and
especially orally administered modified-release (MR) drugs [4]. As with MR
drugs, often the purpose of the liposome is to provide slower drug release
and provide a more prolonged circulatory life of the active drug molecule.
This approach has apparently been successful for Doxil, which may reduce
the cardiotoxicity that is usually associated with doxorubicin [5, 6].
Therefore, many of the concepts used to characterize MR oral formulations
can be adapted to liposomal formulations.
How these concepts are adapted is the subject of considerable
controversy. It is probably no understatement to say that liposomes are
subject to a greater number of factors that can affect product performance
than oral formulations. Small changes in liposome composition such as the
ratio of the lipids, impurities, source of lipids, source of drug substance, and
even the time of year for the same source of lipid, can affect the performance
of the liposomal product [7]. Because there is only limited experience with
these drug products, some aspects of their characterization have not been
finalized. The remainder of this chapter describes what issues are considered
important for liposomal drug characterization, in comparison to tablets or
capsules, and what issues are still evolving.
BIOANALYTICAL ANALYSIS
As with any drug, there is a basic necessity to measure drug concentrations.

The methods used to measure plasma concentrations of the active parent
and/or metabolites from liposomal drugs are essentially the same as those
assays that are used to measure conventional drugs (e.g., HPLC, GC, LC/
MS/MS) [8–10]; there are no significant differences in the analytical
platforms used. Therefore, the development and validation of an assay for a
liposomal drug is same as it is for a more conventional drug.
Characterization of the assay is based on the same elements as an assay for a
conventional drug (e.g., LLOQ, ULOQ, accuracy, precision, etc.).
Therefore, the detailed discussion on analytical method validation in this
edition applies equally to liposomal products [11].
The key difference between liposomal and conventional drugs is the
liposome. Liposomal drugs, once administered to a patient, give rise to at
least two pharmacokinetically/pharmacologically significant species,
namely free drug and encapsulated drug. The measurement of total drug
alone can produce misleading pharmacokinetic characteristics, because
these characteristics are based on both free and encapsulated drugs. This
approach is problematic because it is believed that it is free drug which
mediates activity, and the development of PK/PD relationships with total

Regulatory View of Liposomal Drug Product 527
drug is often unsuccessful in these circumstances. Therefore, it is necessary

to measure both free and total drug concentrations during the development
of these products (total drug concentrations minus free drug concentrations
will equal encapsulated drug concentrations).
The separation of free drug from encapsulated drug is the most critical
step analytically, and it seems to generate the greatest difficulty. The
separation of free and total drug may be problematic, depending on how
fragile the liposome is; the typical methods of separation are sometimes too
harsh for a successful separation. The usual methods include centrifugation
to separate the liposome from free drug (where the supernatant is analyzed),
or some form of filtration (gel filtration, affinity chromatography, etc.) [8].
Double-labeling a liposomal drug with radioactive tracers is a useful way to
distinguish between the liposme and the drug, and it is often done to verify
the suitability of a separation method. However, this method cannot be used
for routine analytical assays because of the need for tracer incorporation,
which typically is not a component of the approved drug product. More
detailed descriptions of liposomal drug separations are available in the
scientific literature.
IN VITRO DRUG RELEASE TEST
In vitro release (IVR) testing is an important component of liposomal drug

characterization. These products are typically administered intravenously,
and might seem to be exempt from bioavailability or bioequivalence
characterization because changes to intravenous formulations generally
only require adequate CMC characterization to be acceptable [12].
However, the liposomes are generally used to modify the pharmacokinetic
and hence the pharmacodynamic behavior of drugs. Therefore, assessing the
characteristic release of the drug from the liposome is crucial. In vitro release
is an in vitro characterization of how the liposomal drug performs with
respect to release of the active drug moiety. The concept of IVR is similar to
a dissolution comparison of oral formulations (tablets and capsules) [3]. In
vitro release represents the final test that assesses the effect of all the
individual chemical characteristics that can affect the drug product
performance. The result serves as a product benchmark, against which
future production batches, modified liposomal formulations, and possibly
generics (if any such entity can be defined) can be evaluated. This evaluation
is important, because IVR is developed with the drug formulation that was
used in the clinical phase 3 trials in which the safety and effectiveness of the
drug product were evaluated. The IVR is the in vitro standard that is used to
assure that production batches of the drug product perform comparably to
the clinical trial formulations, and assures the user that the product will

528 Kumi and Booth
deliver similar effectiveness and safety as that of the phase 3 trials conducted
during product development. In cases where differences are detected, this
finding usually indicates the need for an in vivo bioequivalence study to
determine whether the products actually differ significantly in vivo.
Once a satisfactory IVR test system is established, the amount of drug
released into the solvent is measured as a function of time (see Fig. 1). The
rate and extent of drug release measured by this process is a characteristic of
the drug product and the specific test system. For oral formulations, a
product specification is reviewed and accepted by FDA at the time of drug
approval. For example, Q 80% in 15 minutes for a tablet means that not less
than 80% of the drug is dissolved and in solution within 15 minutes. All
production batches of this tablet are expected to possess this same
performance characteristic. Furthermore, the effect of modifications to the
tablet formulation in terms of dissolution and solubility should be
distinguishable from the original formulation by the dissolution compari-
son. Small insignificant changes should have no effect, whereas important
changes that affect dissolution should be reflected by the dissolution test.
Similar reasoning can be applied to IVR and liposomal drug products.
Therefore, the drug developer can approach the IVR in a similar manner.
First, the test conditions must be established. The test conditions consist of
the apparatus to be used, as well as the solvent, stir rate, temperature,
sampling time, and method of quantification. The goal of this test system is
to distinguish between liposomal formulations that do and do not perform
as acceptably as the reference formulation. The development of this test
system is more difficult than a dissolution test for a conventional tablet or a
capsule. Liposomal performance is sensitive to many seemingly small
influences. Small impurities, differences in the source of liposomal material,
and temperature are a few of the examples that are known to have had a
significant impact on liposomal performance.
For oral formulations, the test system typically consists of a beaker with
solvent that is agitated by a paddle at a given rate (USP method 2) [13] (see
the chapter on dissolution testing in this edition). Alternatively, a basket
rotated at a given rate (USP method 1) is frequently used for capsules [13].
Normally, only some (relatively) minor “tweaking” is necessary before
finalizing a method. The FDA and the USP recognize these methods as the
“state-of-the-art” methodologies; deviating from these generally prescribed
methods requires justification.
However, the development of IVR methods are somewhat more
problematic. The release of drug from the liposome is usually dependent
upon “sink” conditions that are not easily reproduced in vitro. For example,
in the static conditions of a fixed volume of buffer in USP method 2, the
drug concentrations equilibrate because of the lack of “sink” conditions.

FIGURE 1 A typical dissolution profile for a tablet or capsule is shown (upper

panel). An IVR profile for a liposomal drug is shown in the lower panel; Formulations
that release drug too quickly and too slowly demonstrate how the IVR should be
able to distinguish between good and poorly performing formulations.

530 Kumi and Booth
This is probably the single greatest difficulty to overcome. The area of test
apparatus for liposomes IVR is currently an area of considerable research.
Other approaches, such as membrane diffusion, in situ and continuous flow
techniques, have been tested. The continuous flow techniques show
considerable promise, as sink conditions are maintained, but there is no
clear methodological choice yet. The consequence is that, unlike tablet/
capsule dissolution, no standard apparatus is currently available for
liposomes.
In terms of sampling, the drug release should be assessed for a period of
time that is adequate to characterize 80% of the drug release from the
liposome, or until an asymptote is reached [4]. The three batches (two pilot
batches and one small-scale batch) which are used for stability testing
should also be used for IVR development and product specification.
Comparisons should be made using the f2 similarity test, as with dissolution,
which is currently believed to be the appropriate means for comparing
formulations. A difficulty that frequently arises is the time required for the
release of 80% of the drug. This final endpoint is often achieved only after
days of incubation. This time constraint is problematic for routine
monitoring of production lots. Several groups have attempted to address this
problem by accelerated IVR designs. These approaches have incorporated
changes to the method such as increased temperatures or inclusion of
modifiers that accelerate drug release. Although some of these approaches
have successfully increased drug release over a more convenient time frame,
the relation of accelerated release to actual product performance in vivo is
usually unknown. Furthermore, there is currently no consensus on the most
appropriate means for addressing this situation, and it too is another area of
active investigation. Therefore, these situations are typically dealt with on a
case by case basis.
METABOLISM AND PHARMACOKINETICS
Many of the liposomal drug products consist of a previously approved free

drug that is encapsulated in a liposome. Often, it is assumed that the
metabolic and pharmacokinetic behavior of the liposomal drug is the same
as the unencapsulated drug. However, the metabolism and
pharmacokinetics of the liposomal drug may be different compared to the
free drug [14]. Therefore, it is always advisable to evaluate the metabolism
and pharmacokinetics of the active ingredient when encapsulated in
liposomes. These studies should therefore compare, where appropriate, the
absorption, distribution, metabolism, and excretion (ADME) of a liposomal
and nonliposomal drug when

• the two products have the same active moiety,

• the two products are given by the same route of administration,
and
• one of the products is already approved for marketing.
Metabolic characterization should incorporate an in vitro screen (e.g.,

cytochrome P-450 substrate, inhibition and induction, if the free drug is
metabolized by this pathway), and an in vivo study if necessary.
Furthermore, in cases where satisfactory mass balance information is
available for the free drug, then it is feasible to evaluate only the excretion of
the liposomal drug via the major route of elimination. However, if the drug
is not approved in another dosage form, then a full mass balance study
similar to that for any other new molecular entity that delineates the
metabolic pathways and metabolites should be conducted.
It is also important to determine whether encapsulation of an active
ingredient into a liposome alters the volume of distribution (Vd) and
clearance (CL) of the active ingredient. Typically, this alteration of Vd and
CL is the purpose of liposomal encapsulation, but occasionally, liposomes
have been used to enhance drug solubility. Pharmacokinetic studies should
include single-dose and multiple-dose studies that evaluate the
pharmacokinetics of the drug substance after administration of the
liposomal drug product, and a dose proportionality study over the range of
doses that are expected to be used in the patient population.
IN VIVO STABILITY
The stability of a liposome drug product in biological fluid is important for
a safe and effective application of the drug product. It is necessary to
determine that the integrity of the liposome drug product is not
compromized prior to reaching its site of action. Therefore, it is essential
that a bioanalytical method that can distinguish between the encapsulated
and unencapsulated drug (free) product is available (refer to section
Bioanalytical Analysis). Currently, there is considerable discussion as to
what constitutes a stable liposome drug product. The questions that need to
be addressed are
• What amount of drug release is permissible? and

• Is this drug release dependent on the type of liposome and the
intended site of action?
No clear consensus concerning a suitable definition of a stable liposomal

drug product has been reached. However, one possible definition of a stable
liposomal drug product could be that, if over the time course of the in vivo

532 Kumi and Booth
single-dose study, the drug substance remains substantially in the

encapsulated form, and the ratio of unencapsulated to encapsulated drug
substance remains constant; then the liposomal drug product could be
assumed to be stable in vivo [1]. Depending on the intended site of action of
the liposome, when the liposome is stable in vivo, the total drug substance
concentration could be sufficient to determine the pharmacokinetics and
bioavailability of the active pharmaceutical ingredient. However, for an
unstable liposomal drug product, the concentration of both encapsulated
and uncapsulated drug substance should be determined in evaluating the
pharmacokinetics and bioavailability of the drug product [1]. The in vivo
stability of the liposomal drug product will also be influenced by protein
and lipoprotein binding. Hence, the interaction of proteins and lipoproteins
with liposomal drug product should be evaluated.
Drug interaction studies, studies in special populations such as
hepatically and renally impaired patients may have to be conducted
depending on the metabolic fate of the active pharmaceutical ingredient
after encapsulation in liposomes.
BIOAVAILABILITY AND BIOEQUIVALENCE
The important factors in assessing bioavailability and bioequivalence of

liposomal drug products are the release of active moiety from drug product
and the availability at the site of action. Liposomal drug products either act
to deliver drug to a “depot” site from where drug is released slowly into the
systemic circulation and then to its site of action. Alternatively the
liposomes are intended to deliver the drug to a specific site where the drug
acts after release from the liposomal drug product (e.g., tumor uptake of a
liposomal drug). Therefore, depending on the type of liposomal drug
product, a number of questions arise, such as
• Can it be assumed that the plasma drug concentration is an

adequate surrogate for safety and effectiveness of these drug
products?
• Should the lipid moiety be considered as a functional excipient?
• Should it therefore also be required that a reformulated product
or generic product be quantitatively the same as the innovator in
this respect?
• Does the traditional definition of pharmaceutical equivalence
apply to liposomal drug products?
Depending on the intended mechanism of delivery of the active

pharmaceutical ingredient, it may be feasible to conduct bioequivalence

studies using pharmacokinetic parameters as endpoints. The critical

requirement is the availability of a validated, sensitive analytical method
capable of measuring encapsulated and unencapsulated active ingredient.
The liposomal drug product must be stable in vivo. For liposomal drug
products intended to act as a depot and release the drug slowly in the
systemic circulation, it may be feasible to conduct bioequivalence studies
between drug products using pharmacokinetic measures as the endpoints.
For such products, the regulatory criterion that needs to be fulfilled is that
the confidence interval around the ratio of test product to reference product
must fall within 80 to 125% for log-transformed AUC and Cmax.
For liposomal drug products designed to deliver the active ingredient to a
specific site, it may not be feasible to conduct bioequivalence studies using
pharmacokinetic parameters as endpoints. Other methods stipulated in the
CFR for determining bioequivalence, such as comparative clinical safety
and efficacy studies, should be considered as a means of evaluating whether
the liposomal products are therapeutically equivalent. It must be
remembered that these other methods are considered less sensitive in
determining the bioequivalence of the two products. Therefore, the sample
size and the criteria for determining bioequivalence may be more stringent
than a traditional bioequivalence study.
CONCLUSIONS
Generally, the development of liposomal drugs is comparable to traditional

formulations, albeit with the need to address certain liposome-specific
issues. A sensitive specific assay that characterizes free and encapsulated
drug, adequate CMC characterization, and IVR test system development
help direct the in vivo development of a liposomal drug product. Good
biopharmaceutic characterization underpins the clinical pharmacology
characterization of a liposomal drug. Disposition, metabolism, and
excretion of liposomal drugs need to be assessed as new molecular entities,
although the extent of these studies may be abbreviated. Liposomal drug
behavior in special populations may also need to be addressed depending
upon metabolism and excretion studies. Bioequivalence studies for altered
formulations and generics (if possible) can be conducted according to
current practices for free and liposomally encapsulated drugs. It is also
advisable to work with regulatory authorities. Periodic contact with
regulatory authorities during the development of a liposomal drug product
can help to avoid significant differences in expectations regarding the
characterization of the drug at the NDA stage.

534 Kumi and Booth
REFERENCES
1. Draft Guidance for Industry: Liposome Drug Products: Chemistry,

Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability;
and Labeling Documentation; http://www.fda.gov/cder/guidance/index.htm
2. Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally
Administered Drug Products—General, Considerations; http://www.fda.gov/
3. Guidance for Industry: Dissolution Testing of Immediate Release Solid Oral
Dosage Forms; http://www.fda.gov/cder/guidance/index.htm
4. Guidance for Industry: SUPAC-MR: Modified Release Solid Oral Dosage Forms
Scale-Up and Postapproval Changes: Chemistry, Manufacturing, and Controls;
In Vitro Dissolution Testing and In Vivo Bioequivalence Documentation; http://
www.fda.gov/cder/guidance/index.htm
5. Harashima, H.; Iida, S.; Urakami, Y.; Tsuchihashi, M.; Kiwada, H.
Optimization of Antitumor Effect of Liposomally Encapsulated Doxorubicin
based on Simulations by Pharmacokinetic/Pharmacodynamic Modeling. J.
Controlled Release 1999, 61, 93–106.
6. Hussein, M.A.; Wood, L.; His, E.; Srkalovic, G.; Karam, M.A.; Elson, P.;
Bukowski, R.M. A Phase II Trial of Pegylated Liposomal Doxorubicin,
Vincristine and Reduced-Dose Dexamethasone Combination Therapy in Newly
Diagnosed Multiple Myeloma Patients. Cancer 2002, 95, 2160–2168.
7. American Association of Pharmaceutical Scientists meeting. Assuring Quality
and Performance of Sustained Release and Controlled Release Parenterals. April
19–20, 2001. Washington, D.C.
8. Srigritsanapol, A.A.; Chan, K.K. A Rapid Method for the Separation and
Analysis of Leaked and Liposomal Entrapped Phosphoramide Mustard in
Plasma. J. Pharmaceut. Biomed. Analysis 1994, 12, 961–968.
9. Fatouros, D.G.; Hatzidimitriou, K.; Antimisiaris, S.G. Liposomes Encapsulating
Prednisolone and Prednisolone-Cyclodextrin Complexes: Comparison of
Membrane Integrity and Drug Release. Eur. J. Pharmaceut. Sci. 2001, 13,
287–296.
10. Hamilton, A.; Biganzoli, I.; Coleman, R. et al. EORTC 10968: A Phase I Clinical
Trial and Pharmacokinetic Study of Polyethylene Glycol Liposomal
Doxorubicin (Caelyx, Doxil) at a 6-week Interval in Patients with Metastatic
Breast Cancer. Annals Oncology 2002, 13, 910–918.
11. Guidance for Industry: Bioanalytical Method Validation; http://www.fda.gov/
12. The U.S. Code of Federal Regulations, 21 Part 320 Bioavailability and
Bioequivalence Requirements, 2002.
13. Dissolution. 711 U.S. Pharmacopeia, National Formulary 25, NF 20
Supplemental 2002.
14. Bekersky, L; Fielding, R.M.; Dressler, D.F.; Lee, J.W.; Buell, D.N.; Walsh, T.J.
Pharmacokinetics, Excretion and Mass Balance of Liposomal Amphotericin B
(Ambisome) and Amphotericin B Deoxycholate in Humans. Antimicrob. Agents
Chemotherapy 2002, 46, 828–833.

23
Challenges in Drug Development:

Biological Agents of Intentional Use
Andrea Meyerhoff*
INTRODUCTION
The U.S. anthrax outbreak of 2001 has demonstrated the possibility that
biological agents may be used intentionally to cause human disease. This
new awareness underscores the urgency of the public health need for safe
and effective medical countermeasures. Attention to the challenges in the
development of medical countermeasures against biothreat agents can
facilitate their availability. A list of the diseases that can result from the
intentional use of the highest threat biological agents is presented below. It is
followed by a discussion of special issues in drug development presented by
these diseases, and of regulatory mechanisms that can enhance the availability
of such drugs. The chapter concludes with examples of recent regulatory
actions taken by Food and Drug Administration (FDA) to make available
safe and effective drugs for this urgent public health need.
*Current affiliation: Georgetown University, Washington, D.C., U.S.A.
535
536 Meyerhoff
BIOLOGICAL AGENTS AND ASSOCIATED DISEASES

In June 1999, the Centers for Disease Control and Prevention (CDC) convened
a panel of experts to identify the biological agents considered to be of greatest
potential concern. The result was three categories of agents. Because they
cause high mortality or serious illness and are relatively easy to spread, the
organisms in Category A were thought to be of greatest concern. These agents
warrant increased surveillance and the availability of appropriate therapy or
prophylaxis for diseases caused by them [1].
Biological agents-category A (US CDC, June 1999)
ISSUES IN DRUG DEVELOPMENT
The development of efficacy and safety data needed to support the regulatory
approval of a drug for an indication related to the intentional use of a
biological agent raises a number of issues. Many diseases caused by biologic
agents of intentional use rarely occur in nature or are known to contemporary
physicians only by historical reputation. Still others, while continued public
health problems occur in remote areas of the world where the collection of
data and conduct of clinical trials are extremely difficult. It is unethical to
introduce any of the agents into a human population for any purpose,
including the evaluation of drugs.
Up until 2001, there had been 18 cases of naturally occurring inhalational
anthrax reported in the United States, and the events of 2001 resulted in an
additional 11 cases [2]. Inhalational anthrax differs from many other
infections that result from exposure to biothreat agents in that there was a
large outbreak of human disease in Sverdlovsk in the former Soviet Union.
This is thought to have resulted from leak at a military research facility, and
resulted in at least 66 deaths. After several years, an international team of
pathologists published their postmortem findings from these patients, thus
expanding the knowledge of the course of this infection in humans [3]. This
rather sparse database on human disease is one of the most robust for diseases
caused by biothreat agents. Naturally occurring smallpox was declared

Challenges in Drug Development 537
eradicated from the world in 1980, and last seen in the U.S. in 1947 [4]. Few
practicing physicians have seen a case. Pneumonic plague occurs naturally,
but small foci of disease have been found in remote locations that make
systematic study difficult. Intentionally caused disease may differ from what
occurs naturally by a number of variables such as inoculum size, route of
exposure, number of individuals exposed, and rate at which infection may
progress through a population.
There is little regulatory precedent for review of products for such rare
diseases. Even for inhalational anthrax, for which there is some body of data
on human disease, the database is scant when compared with the hundreds
or thousands of patients enrolled in phase III clinical trials of drug evaluation
for more common indications. The need to evaluate drug efficacy for such
diseases can be met in part by the use of animal models. Recent finalization
of the animal efficacy rule [5], which describes the use of animal models for
efficacy evaluation of drugs, represents a new direction in regulatory
approaches to products for use in patients exposed to biothreat agents. The
recognition that there may be scientifically valid animal models from which
drug efficacy information can be derived addresses in part the problems
presented by the need for systematic study for these rare human diseases.
However, access to experimental animals and appropriate laboratory facilities
for such studies can be another limiting factor.
REGULATORY MECHANISMS TO ENHANCE PRODUCT

AVAILABILITY
The development of drugs as countermeasures to bioterrorism present a

number of challenges that heighten the urgency of this public health need. A
number of regulatory mechanisms may be used to address this need. They
are presented below according to stages of product development.
PreIND Meeting
Prior to the submission of an investigational new drug application (IND), a
sponsor may request a preIND meeting with the review division, a means of
opening dialogue with FDA. During this period the sponsor may seek guidance
regarding a wide range of scientific issues, and the preIND meeting offers an
early and systematic way to address them. The process is designed to be
efficient, and permits simultaneous review across all relevant scientific
disciplines. The preIND meeting provides regulatory guidance early in the
development process. It is particularly helpful for drug development that
presents special challenges such as those cited for countermeasures for
bioterrorism. Dialogue can begin at any time during the preIND phase, and

538 Meyerhoff
may address issues including the leveraging of scarce resources such as

experimental animals.
IND Regulations
The IND phase refers to the period that extends from the first use of a
product in human subjects up to the approval for marketing. Prior to
approval, any product may be considered investigational, including those
that are already approved for indications other than that under
development. During this phase, drugs may still be made available for
clinical use. Such use should be consistent with the IND regulations [6].
There are three basic requirements for the IND use of a drug. These are (1)
obtaining informed consent from any patient or subject that receives the
drug, (2) using the product under a protocol of planned use that has been
reviewed by an Institutional Review Board (IRB), and (3) collecting
outcomes data that describe safety and/or efficacy of the investigational
product. FDA has recognized the need to maintain a regulatory standard
of safety and efficacy while meeting the agency’s responsibility to make
medical countermeasures readily available in a public health emergency
such as a release of a biologic agent. In this regard, sponsors such as federal
or local public health agencies may make use of a “streamlined IND” or
“contingency protocol” that adheres to regulatory requirements while
meeting emergent need. Such applications may be appropriate to a
population exposed to a biological agent.
NDA Regulations
The new drug application (NDA) regulations describe the standards of drug
approval for marketing. Within the NDA regulations are certain provisions
that can enhance availability of medical countermeasures against biothreat
agents. These include the accelerated approval regulations and the animal
efficacy rule.
The accelerated approval regulations [7] describe the use of a surrogate
marker of efficacy thought reasonably likely to offer a benefit of decreased
serious morbidity or mortality. The regulations require the collection of
postmarketing information to validate the choice of surrogate. Such markers
have been used for other classes of drugs such as the antiretrovirals, where
the CD4 count was considered a surrogate marker. The accelerated approval
regulations were the basis of the FDA approval of the first antimicrobial for
an indication related to a biological agent of intentional use, ciprofloxacin
for postexposure inhalational anthrax. A more detailed discussion of that
approval is presented below.

Finalized in May 2002, the animal efficacy rule [5] may apply to the
study of that a disease cannot be studied in humans because it is extremely
rare and/or unethical to introduce the disease into a human population. In
such a case, this regulation describes the development of efficacy data in a
scientifically valid animal model of the diseases of interest. The animal rule
applies only to efficacy data; safety data for any drug evaluated in this manner
would be expected to be developed in a human population. A product
approval based on the animal rule would also require the collection of
outcomes data in the postmarketing period.
Priority Review of New Drug Application

At the time of the NDA submission, drug availability may also be accelerated
by a priority review. This is a request made by the drug sponsor at the time
of submission, and is generally used for products of special public health
significance. Priority review status shortens the time of NDA review to six
months.
RECENT REGULATORY ACTIONS ON DRUGS FOR BT/BW

INDICATIONS
Ciprofloxacin for PostExposure Inhalational Anthrax
In August 2000, the U.S. Food and Drug Administration (FDA) approved
Cipro® (ciprofloxacin hydrochloride) for postexposure inhalational anthrax.
This was the first antimicrobial drug approved by FDA for use in an infection
due to a biological agent of intentional use.
The study of ciprofloxacin for prevention of inhalational anthrax was
performed in a nonhuman primate model, the rhesus macaque. It was
planned and conducted by investigators at the U.S. Army Medical Research
Institute of Infectious Diseases (USAMRIID) in 1990 at the start of the
war in the Persian Gulf. The results demonstrated a significantly improved
survival rate for animals that received ciprofloxacin following exposure to
aerosolized B. anthracis compared to animals that received no antimicrobial.
Ciprofloxacin serum concentrations were measured in these animals, and
it has been shown that these levels are reached or exceeded in various
human populations that receive ciprofloxacin in the doses recommended
for this indication. Human serum concentrations could also be correlated
with clinical outcome when viewed in the context of in vitro drug
susceptibility of B. anthracis.

540 Meyerhoff
Ciprofloxacin serum concentrations in humans served as a surrogate

endpoint for the efficacy of ciprofloxacin in postexposure inhalational
anthrax. As such, the efficacy data in the Cipro® application met the criteria
for approval under the accelerated approval regulations.
Since the 1940s, studies of inhalational anthrax had been undertaken in a
number of animal species, many in the rhesus macaque. The study of human
disease resulting from sporadic industrial exposure and from the 1979
outbreak in Sverdlovsk provided an understanding of inhalational anthrax
that demonstrated that the macaque is a relevant animal model of this disease.
The applicability of this model was based on data attesting to the similarities
in pathogenesis, clinical course, and tissue pathology in rhesus monkeys and
humans with inhalational anthrax. Ciprofloxacin had been used widely and
has a well-characterized safety profile. There also existed a significant body
of pediatric safety data such that the indication was approved for pediatric
use as well.
The availability of a suitable animal model for inhalational anthrax, the
demonstration of a significant survival advantage in experimental animals
that received ciprofloxacin, the use of ciprofloxacin serum concentrations in
humans as a surrogate endpoint, the well-established body of safety data for
this drug, and the unanimous concurrence of the Anti-Infective Advisory
Committee constituted the scientific basis for this approval [8].
Doxycycline and Penicillin for PostExposure Inhalational Anthrax

In November 2001, FDA further expanded the options for the management
of patients exposed to aerosolized anthrax spores with the publication of a
Federal Register (FR) notice providing scientific data and dosing
recommendations for two other drugs already approved for anthrax,
doxycycline and penicillin [9]. At the beginning of the U.S. anthrax outbreak
of fall 2001, the FDA Center for Drug Evaluation and Research (CDER)
recognized the need to expand options for the management of individuals
exposed to spores of B. anthracis. At that time, there were products in the
penicillin and tetracycline classes that were already approved for treatment
of anthrax in general, but did not include specific dosing recommendations
for postexposure management in the label.
It was also recognized that the USAMRIID animal model of postexposure
inhalational anthrax that supported the approval of cipro-floxacin also
included cohorts that received doxycycline or penicillin. Both of these drugs,
for which there are both innovator and generic products, had been approved
for decades and both were characterized by a substantial safety database.
Review of pertinent pharmacokinetic and safety data for these drugs suggested
that sufficient scientific evidence existed to support the publication of dosing
recommendations for these two drugs for the management of individuals

exposed to aerosolized B. anthracis. This information was made available to

the public as an FR notice in November 2001, with a simultaneous request
for manufacturers of these products to submit labeling supplements to FDA
such that this indication and dosing information could be added to the
package insert [9].
CONCLUSION
The threat of the intentional use of biological agents presents an urgent public
health need. Recognition of the agents of highest threat, the challenges
presented by the development of drugs to counter these threats, and the
regulatory mechanisms to enhance the availability of such drugs are important
tools in our biodefense preparedness.
REFERENCES
1. Rotz, L.; Khan, A.S.; Lillibridge, S.R., et al. Emerging Infectious Diseases 2002,
8. Available from http://www.cdc.gov/ncidod/eid/vol8no2/01–0164.htm
2. CDC. Update: Investigation of Bioterrorism-related Inhalational Anthrax—
Connecticut, 2001. MMWR 2001;50:1049–51.
3. Abramova, F.A.; Grinberg, L.M.; Yampolskaya, O.V.; Walker, D.H. Pathology
of Inhalational Anthrax in 42 Cases from the Sverdlovsk Outbreak of 1979.
Proc. Natl. Acad. Sci. USA. 1991, 90, 2291–2294.
4. CDC. Eradication: Lessons from the past. MMWR 1999, 48 (SU01), 161.
5. U.S. Food and Drug Administration. New Drug and Biologic Products; Evidence
Needed to Demonstrate Effectiveness of New Drugs When Human Efficacy
Studies Are Not Ethical or Feasible. Federal Register 2002, 67, 37988–37998.
6. Code of Federal Regulations: Investigational New Drug Application, 21 C.F.R.
Sect. 312.1–160(2002).
7. Code of Federal Regulations: Subpart H-Accelerated Approval of New Drugs
for Serious or Life-Threatening Illnesses, 21 C.F.R. Sect. 314.500–560 (2002).
8. Anti-Infective Drugs Advisory Committee to the Food and Drug Administration,
meeting of July 28, 2000, to consider Supplemental New Drug Applications 19–
537/S038, 19–847/S024, 19–857/S027, 19–858/S021, 20–780/S008 for Cipro®
(ciprofloxacin). Agenda, briefing materials, roster, slides and transcript available
at: http://www.fda.gov/ohrms/dockets/ac/cder00.htm. Accessed May 14, 2002.
9. Prescription Drug Products; Doxycycline and Penicillin G Procaine Administration
for Inhalational Anthrax (Post-Exposure). Federal Register 2001, 66, 55679–
55682. Available at: http://www.fda.gov/cder/drug/infopage/penG_doxy/
default.htm. Accessed May 14, 2002.

24
The Regulation of Antidotes for Nerve

Agent Poisoning
Russell Katz and Barry Rosloff

On February 5, 2003, the U.S. Food and Drug Administration (FDA)

approved a New Drug Application (NDA) for the use of pyridostigmine
bromide as a pretreatment for poisoning with the nerve agent, soman. This
approval was granted under recently adopted regulations that permit the
marketing of such treatments on the basis of effectiveness data obtained in
animal studies. This chapter will discuss the regulatory and scientific issues
raised by these applications generally, as well as those considered for this
specific application.
The regulatory and scientific questions raised in the consideration of the
standards that must be met by a sponsor wishing to market a treatment for
individuals exposed to poisoning by nerve agents are complex and novel. In
this chapter, these questions will be identified, and potential answers discussed,
in the context of a proposed treatment for poisoning with the nerve agent,
soman. While the chapter will be concerned with this specific example, most
of the issues raised will be relevant to the consideration of the standards to
543
544 Katz and Rosloff
be applied to any application for a proposed treatment for nerve agent

poisoning.
REGULATORY ISSUES
In order to understand the regulatory issues that are unique to a consideration

of applications for treatments for nerve agent poisoning, it is imperative to
have an understanding of the legal standards that must be met by any
application for marketing of a new drug. In this chapter, we will focus almost
exclusively on the effectiveness standard; while the law also requires that a
drug be shown to be safe in use, we will not specifically discuss this
requirement.
The Federal Food, Drug, and Cosmetic Act (the Act), the statutory basis
for drug approval in the United States, sets out the requirements that must
be met before an application for a drug product may be approved. Among
other things, the Act requires that a sponsor provide “substantial evidence”
of effectiveness that the drug will have the effect described in product
labeling. The Act itself provides a definition of substantial evidence as
follows:
…“substantial evidence” means evidence consisting of adequate and

well-controlled investigations, including clinical investigations, by
experts qualified by scientific training and experience to evaluate the
effectiveness of the drug involved, on the basis of which it could fairly
and responsibly be concluded by such experts that the drug will have
the effect it purports or is represented to have under the conditions of
use prescribed, recommended, or suggested in the labeling or proposed
labeling thereof [1].
The critical portion of the definition for our purposes is the requirement for
clinical investigations with the drug. The word clinical has traditionally been
interpreted to mean human; that is, the Act has traditionally been interpreted
to require that a drug be shown to be effective in humans before it may be
approved for human use.
Typically, clinical trials that have served as the adequate and well-controlled
trials on which approval has been based have demonstrated an effect of the
proposed treatment on a relevant measure of clinical performance. For
example, drugs to treat patients with seizures are approved on the basis of a
showing that they decrease the number of seizures compared to a control
group. Similarly, drugs to treat patients with Major Depressive Disorder are
approved on the basis of the drug’s beneficial effect on a scale that assesses
the patient’s depressive symptoms compared to a control group. Almost all

Regulation of Antidotes for Nerve Agent Poisoning 545
drugs are approved on the basis of a beneficial effect on a symptom or sign

that is of obvious relevance to the patient’s clinical status.
However, some drugs have been approved on the basis of a drug’s beneficial
effect on a measure that is not immediately obviously relevant to how the
patient feels or to the patient’s functioning. These measures are called
“surrogate markers,” and in those cases in which approval has been based
on a beneficial effect on such a surrogate marker, the approval has been
based on the Agency’s finding that the effect on the surrogate can be taken to
imply an effect on a clinical outcome of importance.
For example, the Agency has long approved drugs proposed as treatments
for hypertension on the basis of a beneficial effect on blood pressure. Blood
pressure is a surrogate marker, because it is a measurement that, in and of
itself, is not directly tied to the patient’s clinical status or symptoms (unless,
of course, it is very low or very high). Another example is the class of
cholesterol lowering agents. These drugs are approved on the basis of a
beneficial effect on serum cholesterol, a laboratory test that is not directly
linked to the patient’s clinical status at the time of the test. In both of these
examples, the Agency has approved treatments because lowering blood
pressure (in patients with hypertension) and lowering cholesterol (in patients
with elevated cholesterol) have been shown, over time, to result in a decrease
in negative clinical outcomes (strokes, heart attacks, etc.). The value of basing
approval in these (and other) cases on an effect on a surrogate is that trials
designed to assess the important clinical outcomes (stroke, death, etc.) would
need to be of extremely long duration, making them essentially impossible
to perform adequately.
In 1992, the regulations (those rules promulgated to interpret the provisions
of the Act) were amended to explicitly permit the approval of drugs that
have an effect on a surrogate marker that had not been shown to definitively
produce a clinical benefit. The new provisions, referred to as Subpart H of
the regulations, define the conditions under which such an approval may be
granted as follows:
…on the basis of adequate and well-controlled clinical trials establishing

that the drug product has an effect on a surrogate endpoint that is
reasonably likely, based on epidemiologic, therapeutic,
pathophysiologic, or other evidence, to predict clinical benefit [2].
In 1997, the Act itself was amended to include this specific standard as a
basis for approval.
Prior to the 1992 change in the regulations, drugs that were approved
based on their effects on surrogate markers were approved on the basis of an
effect on surrogate markers that were considered to have been “validated”;
that is, proven to predict an actual clinical benefit (as in the case of

antihypertensives and cholesterol lowering agents). After the 1992

amendments to the regulations, however, the Agency could approve a drug
on the basis of an effect on an “unvalidated” surrogate marker; that is,
approval could be granted on the basis of an effect on a measurement that
had not yet been demonstrated to predict a beneficial effect on a clinical
outcome. The amendment did, however, require that the clinical effect of
interest be shown in studies completed after approval (and, indeed, it was
expected that the studies designed to demonstrate this effect would be going
on at the time of approval) [3].
It is important to note that this new provision still required that the showing
of the effect on a surrogate marker be made in humans; that is, while in
some sense the new requirement could be seen as permitting a “lower”
standard of effectiveness to be met in certain circumstances (because an effect
on a clinically meaningful outcome need not be shown), this provision did
not dispense with the requirement in the Act for a finding in “clinical
investigations,” that is, the drug must be shown to have a beneficial effect
on the surrogate marker in humans. While there has been some discussion
about whether or not the source of the evidence on which the conclusion
that the proposed surrogate marker is considered reasonably likely to predict
the clinical benefit can be exclusively derived in animals, the general view is
that it can. However, the effect on the surrogate must be, under the new
provisions, shown in humans.
Despite this new standard of approval having been incorporated into the
law, the Agency felt that there might be situations in which even this standard
might be inadequate to support the approval of certain other products, namely
products intended to treat patients who had been the victims of various
types of poisonings. Specifically, it was felt that it was important to permit
the approval of treatments for these patients, but that adequate and well-
controlled studies in humans were not feasible for ethical reasons. That is, it
was generally considered unethical to perform studies designed to demonstrate
the effectiveness of an antidote to poisoning, because such studies would
require that subjects be purposefully exposed to the poison. Given this state
of affairs, the Agency adopted regulations that set out the evidence that the
Agency might rely upon when considering the approval of applications for
proposed antidotes to poisons.
These regulations, referred to as Subpart I and published in the Federal
Register on May 31, 2002, set out the following requirements:
1. The proposed treatment is intended to ameliorate or prevent

“serious or life-threatening conditions.”
2. The approval may be based on adequate and well-controlled
animal trials.

3. The results of these studies must be reasonably likely to predict

benefit in humans.
4. Studies in animals will be relied upon only where:
a. There is a reasonably well-understood pathophysiological
mechanism of the toxicity of the poison and its prevention or
substantial reduction by the drug.
b. The effect is shown in multiple animal species, or a single
species expected to react in a manner predictive of how
humans will respond.
c. The endpoint in the animal studies is clearly related to the
desired outcome in humans, usually mortality or an effect on
major morbidity.
d. Data on the kinetics and pharmacodynamics of the drug, as
well as other relevant data, allow the selection of an
appropriate dose in humans [4].
It is instructive to further examine these requirements.

First, it is important to note that the regulations explicitly state that they
do not apply in those cases in which already existing provisions could be the
basis for approval (e.g., subpart H in those cases, for example, in which
approval could be based on a drug’s effect on a surrogate marker in humans,
etc.). This explicit statement embodies the Agency’s acknowledgment that
the proposed reliance on the results of animal studies, while justifiable and
nonviolative of the Act’s requirements, should only be reserved for
extraordinary circumstances [5].
That the regulations propose a unique approach to drug approval is clear,
but some discussion is worthwhile to illuminate some of the fundamental
differences underlying this approach and current practice and standards of
drug approval.
The notion that a drug may be approved for marketing in humans on the
basis of data in nonhuman species highlights an important concept routinely
applied in current drug approval.
Ordinarily, a drug is approved for marketing on the basis of an empirical
demonstration of benefit on an outcome that is considered self-evidently
meaningful to the patients (or, less frequently, as we have seen, on a
surrogate measure that predicts such an effect). Critically, the presumed
mechanism of action of the drug, while of interest and even importance
in certain regards, is of little regulatory concern. That is, a detailed
understanding of how the drug produces the effect of interest is not
required for drug approval, in the typical case. A sponsor is required to
show that the drug is effective (appropriately defined), but is not required
to prove the mechanism of its effect. Indeed, it is fair to state that we

have a complete understanding of the mechanism of action of perhaps

only a tiny fraction of currently approved drugs, but the Act does ensure
that they have been shown to be effective. Were the Agency to require
that a sponsor identify a drug’s mechanism of effect prior to approval,
few drugs would ever reach the market. The reasons for not requiring a
sponsor to document the mechanism of action of a drug prior to its
approval are clear: the pathophysiology of most diseases is not completely
understood, and therefore it is irrational to expect that all of a drug’s
relevant actions can be identified at any given time. Further, our current
understanding of a disease’s pathophysiology and a drug’s actions may,
ultimately, turn out to be incorrect, and it would be inappropriate to
base the approval of a drug product, even in part, on such an incorrect
understanding. The law’s requirement that the drug be shown, empirically,
to be effective, is the most appropriate effectiveness standard that can be
applied.
Similarly, typically, current drug approval attempts to rely on the fewest
possible assumptions about other aspects of a drug’s effects. The Agency,
again, ordinarily relies upon an empirical demonstration of effectiveness as
provided by data from clinical studies that are adequate and well-controlled
(i.e., appropriately designed and conducted), rather than relying on
assumptions about underlying pathophysiologic events, presumed mechanism
of action of the drug, etc. For example, the requirement for a concurrent
placebo control group (where appropriate), rather than a reliance upon
assumptions about patients’ responses in the absence of treatment, embodies
the Agency’s preference for an empirical showing of a drug’s effectiveness.
Many other aspects of adequate trial design incorporate the need for an
empirical showing, rather than an assumption-based conclusion, of
effectiveness. As a general principle, if data can be adduced to answer a
specific question, this is to be preferred to relying upon assumption-based
approaches.
As can be seen from an examination of Subpart I, however, while there
is a requirement for the generation of evidence (in animals, for example,
the requirement that the drug’s effect be shown in multiple animal species),
the rule permits a drug to be approved on the basis of a number of
(ordinarily untestable) assumptions. Specifically, the requirement that the
pathophysiology of the poison-induced toxicity and the drug’s mechanism
of its amelioration be well understood elevates to a primary position a
consideration that is, as explained above, ordinarily a matter of little
regulatory import. Further, the over-arching principle on which the
proposed rule is fundamentally based, the ability to extrapolate from
data in animals to conclusions about a drug’s effects in humans, must
ultimately be seen as an assumption that would ordinarily be considered
unprovable. Indeed, the provisions of the rule, as outlined above, exist to

provide the maximum reassurance that the results seen in animals will
apply to humans. Ultimately, however, this evidence can only provide a
reasonable likelihood that this is true; it would ordinarily not be expected
to provide proof.
Nonetheless, given the limitations (essentially impossibility) of performing
adequate and well-controlled trials of proposed antidotes in patients who
have been exposed to deadly toxins, and given the desire to develop and
make these products available, under an approved NDA, the requirements
of Subpart I are comprehensive and appropriate, with the caveats expressed
above.
Given this background of the relevant regulatory issues, mechanisms, and
concerns, it will be illustrative to examine these issues as they relate to the
development of one potential treatment, pyridostigmine, for the treatment
of intoxication with one specific nerve agent, soman.
SCIENTIFIC ISSUES
A chemical agent has been defined by the North Atlantic Treaty Organization
as, “…a chemical substance intended for use in military operations to kill,
seriously injure or incapacitate people because of its physiological effects.”
Various of these weapons have been used throughout the 20th century (e.g.,
mustard gas in World War I, nerve gas in Iraq in the 1980s, etc.) [6, 7]. Here,
however, we will concentrate on the development of treatments for
intoxication with nerve agents, specifically Soman.
Nerve agents are all members of the class of organophosphate compounds,
in which class are also included various available pesticides. Nerve agents
were first synthesized in Germany before World War II, and include tabun,
sarin, cyclosarin, and soman. These agents are liquid and volatile at room
temperature, and can enter the body via inhalation and directly through the
skin [6].
The primary action of these agents is to phosphorylate
acetylcholinesterase (AChE), and thereby irreversibly inactivate it.
Acetylcholinesterase is the primary enzyme responsible for hydrolyzing
acetylcholine (the primary neurotransmitter at nicotinic and muscarinic
receptors), so the net effect of poisoning with nerve agents is an
accumulation of acetylcholine at these receptors. Excessive accumulation
of acetylcholine at these receptors gives rise to a number of signs and
symptoms, depending, of course, on the degree of such accumulation.
Symptoms can range from excessive bronchial secretions, rhinorrhea, miosis,
blurred vision, abdominal cramping, increased salivation, sweating, and
lacrimation, urinary frequency and involuntary urination and/or defecation,
and can progress to vomiting, bradycardia, generalized muscle weakness,

paralysis, pulmonary edema, hypotension, respiratory depression, seizures,

coma, and death [7, 8].
While the nerve agents ultimately bind irreversibly to the AChE the nerve
agent-AChE complex can be uncoupled by treatment with oximes (such as
pralidoxime), but only within a specific period of time, unique to the specific
nerve agent. After this period of time, the binding is irreversible. This time-
related irreversibility of binding is referred to as “aging” [9].
Several treatments are currently approved for the treatment of
organophosphate pesticide poisoning. Specifically, atropine and pralidoxime
are approved for the management of patients who have suffered a toxic
exposure to organophosphorous or carbamate insecticides. Atropine is a
competitive inhibitor of acetylcholine at muscarinic receptors, and can treat
the hypersecretion, intestinal cramping, and bronchoconstriction induced
by nerve agents.
Pralidoxime is an oxime; as described above it can “reactivate” AChE by
splitting apart the nerve agent-AChE complex, thereby regenerating AChE,
making it available to hydrolyze acetylcholine at the synapse. As noted above,
if sufficient time has passed before the nerve agent-AChE complex is exposed
to pralidoxime, the binding becomes irreversible. In the case of soman, this
aging process is extremely rapid (several minutes), and thus pralidoxime
alone is not considered to be helpful for treating poisoning with this agent
[9].
Pyridostigmine, a reversible inhibitor of AChE, with poor penetrance
into the central nervous system, which is approved for patients with
myasthenia gravis, has been proposed as a treatment for prevention of
mortality in patients exposed to nerve agents, in particular soman, in
combination with acute treatment with atropine and pralidoxime.
Pyridostigmine is not proposed as an acute treatment for soman poisoning;
rather, it is proposed as a prophylactic treatment. In theory, pyridostigmine,
given in appropriate amounts and at appropriate times, protects the
organism by reversibly binding with (some) AChE, preventing the
irreversible binding of these AChE molecules with the nerve agent. In time,
the AChE-pyridostigmine complex will spontaneously dissociate, and a
critical amount of AChE will be available to hydrolyze acetylcholine at the
receptor (if the exposure to the nerve agent has been transient). In this
scenario, atropine and pralidoxime are still considered necessary for
pyridostigmine to be effective [7, 8, 10].
Given these basic facts, it is instructive to examine the evidence available
and the issues raised when applying the Agency’s proposed criteria for
approval of antidotes to the case of pyridostigmine as a proposed treatment
for intoxication with soman.

EVIDENCE OF PYRIDOSTIGMINE’S EFFECTIVENESS IN

ANIMALS
First, it is important to briefly describe the evidence on which is based the

claim that pyridostigmine protects against soman-induced lethality.
Studies have demonstrated that when monkeys are pretreated with
pyridostigmine, then exposed to soman, and then treated with atropine
and pralidoxime, they have significantly decreased mortality compared to
monkeys similarly treated with atropine and pralidoxime, but not pretreated
with pyridostigmine. The effect of the treatment regimen is assessed by an
examination of the Protective Ratio (PR), defined as the ratio of the LD50
(the dose of nerve agent required to kill 50% of the animals) after
pretreatment with pyridostigmine to the LD50 without pretreatment with
pyridostigmine. In monkeys, PRs of >40 have been seen after pretreatment
with pyridostigmine, suggesting a large effect of pyridostigmine
pretreatment on soman-induced lethality. In guinea pigs, PRs after
pretreatment with pyridostigmine were about four times those seen without
pretreatment with pyridostigmine, but no such marked increases in PRs
with pretreatment compared to those without pretreatment were seen in
mice, rats, or rabbits [9].
Explanations of Pyridostigmine’s Differential Effect Across

Species
Because reliance on animal studies for drug approval presupposes a
consistent finding of the treatment across multiple animal species, the lack
of a consistent finding across species requires an explanation to justify that
the species in which the beneficial finding is seen are more relevant to
humans.
One proposed explanation for the differences seen in degree of protection
of the various species relates to the view that relative rates of
decarbamylation of AChE after carbamylation by pyridostigmine determine
the species-specific sensitivities to pyridostigmine, and that the relatively
rapid rate of decarbamylation in monkeys, the species in which
pyridostigmine is most effective, is closer to that of humans than to other
species (the mechanism of pyridostigmine-induced protection is believed
to be carbamylation of the active site of AChE; subsequent decarbamylation
must occur in order for the enzyme to be functional). Several articles in the
literature present results of studies purporting to compare the rates of
decarbamylation in various species, but the results are fairly limited, and
not all studies documented such differences [11–13]. Further, these studies
only evaluated the activity of the enzyme in plasma and red blood cells
(RBC), but provide no assurance that relevant species differences are seen

at the sites of action that would presumably be relevant for protection in

humans (e.g., the neuromuscular junction). In addition, these studies did
not examine enzyme regeneration rates in vivo, where concentrations of
pyridostigmine might be expected to be more complex (e.g., varying over
time) than in these assays, where pyridostigmine concentrations were held
relatively constant. Finally, even if a correlation could be shown between
rate of decarbamylation and sensitivity of species to pyridostigmine
protection (or lack of protection), this does not establish that this is a
mechanism that is operative in determining protection. It has even been
postulated that if decarbamylation is too fast, this might result in a loss of
effectiveness, because this might result in AChE that is available for
inhibition by soman, if it is still present in sufficient quantities.
Another explanation for species differences in sensitivity to pyridostigmine-
induced protection from soman toxicity that has been proposed relates to
species differences in carboxylesterase activity.
This enzyme is considered to be important in the detoxification of soman
in those species in which it exists. It has been shown that monkeys and
humans have little to no carboxylesterase activity, and therefore, if
carboxylesterase activity is indeed an important determinant of
pyridostigmine-induced protection, it has been postulated that these two
species would be expected to respond similarly to pyridostigmine, in contrast
to other species which have higher levels of carboxylesterase activity and do
not respond well to pyridostigmine.
This hypothesis has been examined in guinea pig, rat, mouse, and rabbit.
Appropriate protection was seen, and the degree of protection was much
more similar, and greater, in the presence of a carboxylesterase inhibitor
(which “created” species that were, in theory, similar in their degree of
carboxylesterase activity to humans and monkeys) [14, 15].
However, a number of questions regarding the role of carboxylesterase in
determining species-specific sensitivity to pyridostigmine arise.
For example, it is not immediately obvious, in theory, why the degree of
carboxylesterase activity should be a determinant of the efficacy of
pyridostigmine. Specifically, carboxylesterase decreases the plasma levels
of soman, but it should not, theoretically, affect the plasma levels of soman
associated with lethality (although the dose of soman necessary to be given
to achieve the level associated with lethality should be greater in species
with high carboxylesterase activity compared to those with less activity). If
this is true, the protective ratio (the ratio of the doses of soman needed to
produce an LD50 with and without pyridostigmine pretreatment) should
not change. For example, if a species has twice as much carboxylesterase
activity as another species, the dose of soman needed to produce the LD50
in the former will be twice as great as in the latter, in both pyridostigmine-
treated, and nonpyridostigmine-treated animals, thereby yielding the same

protective ratio in both species, all other things being equal. On the other
hand, it has been hypothesized that, in species with high carboxylesterase
activity, the ability of carboxylesterase to eliminate soman becomes
saturated with increasing soman doses such that plasma levels of soman
increase in a nonlinear fashion (i.e., relatively low levels are achieved with
doses below the saturation point). In this case, pyridostigmine, even if it
had activity in these species, would not significantly increase the protective
ratio. (One way of conceptualizing this is that pyridostigmine can increase
the LD 50 of soman in all species, but that it is difficult to show an effect
on the protective ratio in species which are already protected by an
intrinsically high carboxylesterase activity.)
It is also possible that the carboxylesterase inhibitor given in the studies
noted above has additional actions that could explain the results. Monkeys
were not used in these studies; a lack of effect of the inhibitor on the efficacy
of pyridostigmine in this species, which has low carboxylesterase activity,
would support the conclusion that the inhibitor potentiated pyridostigmine
in the other species by inhibiting carboxylesterase.
In addition to these caveats, it is critical to note that additional
mechanisms, aside from inhibition of AChE, may be involved in soman-
induced toxicity. For example, recent articles in the literature implicate the
NMDA receptor complex as being important in the production of nerve
agent-induced seizures [16–18]; other articles document the effects of
soman-induced intoxication on brain levels of GABA-ergic, dopaminergic,
and cholinergic systems, as well as on IL-1beta levels in rat brain [19].
These investigations suggest the complex number of systems that may
mediate soman poisoning, and the complex time-concentration relationships
that occur between levels of a host of chemical species (endogenous species
and soman) that result in soman-induced injury, and pyridostigmine-induced
prevention of injury, all of which may vary among species. While these
studies discuss mechanisms of soman-induced brain injury in various species,
and pyridostigmine is considered not to cross the blood-brain barrier, they
may seem irrelevant to the question of pyridostigmine’s effectiveness.
However, there is evidence that pyridostigmine does have central effects,
thereby raising additional questions about how well the mechanisms of
pyridostigmine-induced protection are understood.
In addition, there may be other actions of pyridostigmine, aside from
inhibition of AChE, which may contribute to its ability to protect (in animals)
against nerve agent toxicity, including alternate (though currently
unrecognized) mechanisms that might diminish acetylcholine activity at the
neuromuscular junction. Indeed, it is fair to say that the mechanism of action
of pyridostigmine as a pretreatment for soman-induced toxicity may not be
completely understood, making it impossible to conclude with certainty that
(1) the protection it confers on monkeys (and to a lesser extent guinea pigs)

will be seen in humans, and (2) monkeys represent the most relevant model
for human responsiveness.
DOSE CONSIDERATIONS AND DIFFICULTIES IN THE

INTERPRETATION OF DRUG EFFECT ON SURROGATE
MARKERS
A further critical criterion for relying on animal studies to support a conclusion

about the effectiveness of an antidote in humans is that the animal studies
must provide a basis for identifying a dose of the antidote in humans that
will be effective.
In the case of pyridostigmine pretreatment against soman-induced toxicity,
the dose necessary to produce inhibition of cholinesterase in red blood cells
(RBC) of between 20–40% has been proposed as the appropriate dose. The
Department of Defense has shown that a dose, in humans, of pyridostigmine
of 30 mg every eight hours will result in this degree of RBC cholinesterase
inhibition throughout most of the dosing interval.
The degree of RBC cholinesterase inhibition as a guide to appropriate
dosing in humans had been proposed as a surrogate marker of activity, as
defined earlier. That is, it had been proposed that when RBC cholinesterase
is inhibited between 20–40%, humans will be protected from soman-induced
toxicity [9]. Because the true clinical endpoint (mortality) cannot be studied
in adequate clinical studies, the achievement of the desired degree of RBC
cholinesterase inhibition had been proposed as a surrogate for the clinical
endpoint of interest.
While this surrogate cannot be validated in humans (i.e., we cannot know,
definitively, in humans, if this prediction of protection is accurate), the first
step in accepting RBC cholinesterase as a useful surrogate in humans would
be to validate its predictive effect in animals. That is (because the experiment
can be done in animals), it should be theoretically possible to validate in
animals that the degree of RBC inhibition proposed as protective in humans
is, in fact, predictive of protection in animals.
Experiments have been performed in animals that allow an evaluation of
the validity of RBC cholinesterase inhibition with pyridostigmine pretreatment
as a surrogate for survival. These experiments measured pyridostigmine-
induced RBC cholinesterase inhibition and protection against soman lethality
following a range of pyridostigmine doses. The showing of a correlation
between enzyme inhibition and survival would give credence to (though would
not constitute proof of) the idea that pyridostigmine-induced RBC
cholinesterase inhibition is an appropriate choice for a surrogate in humans.
The results of these experiments, however, in general demonstrated no
correlation between pyridostigmine-induced RBC cholinesterase inhibition

and survival. In particular, the monkey studies showed that the effect on
survival when the degree of RBC cholinesterase inhibition was 20–40%
was equivalent to the effect on survival when the degree of RBC inhibition
was essentially equal to that in the control group. This finding strongly
suggests that the increase in survival associated with pyridostigmine
pretreatment is not directly related to the degree of RBC cholinesterase
inhibition. If this is true, choosing a dose that will ensure protection in
humans based on achieving a particular degree of RBC inhibition in humans
is not supportable, because it is not a valid surrogate (in animals); that is,
the degree of RBC inhibition does not predict the outcome of interest
(increased survival) [9].
Even if such a correlation between RBC cholinesterase inhibition and
increased survival in the animal had been demonstrated, it might still be a
misleading surrogate, because we do not know the relationship (in animals,
or, of course, in humans) between the degree of RBC cholinesterase inhibition
and cholinesterase inhibition (if cholinesterase inhibition is relevant at all) at
the site of action presumably responsible for the protection (e.g., the
neuromuscular junction).
Indeed, closer consideration suggests that RBC cholinesterase inhibition
is, a priori, likely to be an inadequate surrogate (even if cholinesterase
inhibition is a relevant mechanism). RBC cholinesterase inhibition is, in
this case, likely to be merely reflective of the plasma level of pyridostigmine.
However, surrogates are more likely to be “valid” the more they reflect
biological processes that are occurring as close as possible to the final “step”
in the pathophysiologic chain of events. This is because there may be many
events leading to the production of the symptoms of concern. The more
the surrogate is reflective of events in the final “steps” of disease production,
the more likely an effect on the surrogate will represent an effect on the
symptoms of interest; that is, in such a case, it is presumed that the desired
drug effect is mediated through the surrogate, “ensuring” that the effect
will be seen on the clinical symptoms (it is for this reason that a detailed
understanding of the pathophysiology of the disease, and a detailed
understanding of the mechanism of action of the drug, contribute to
increased confidence that the drug’s effect on the surrogate will have the
desired effect on the disease; as noted above, of course, such complete
knowledge is usually lacking). The further removed from the final “step”
the processes measured by the surrogate are, the greater the possibility
that the drug’s effect is on a pathway that is not (entirely or at all) related
to the ultimate outcome of interest. Observing an effect on the “final”
pathophysiologic event(s) helps to increase the likelihood that the surrogate
actually reflects the steps in the biological processes that are critical to the
production of the outcome. It is critical to note, however, that even in the
best case (that is, one in which the relevant mechanism of action of the

drug and the relevant pathophysiologic events are considered to be

understood), a correlation of the effect on the surrogate with the clinical
outcome of interest cannot be considered proof that the effect on the
surrogate must predict the desired clinical outcome [20].
In any event, RBC cholinesterase inhibition fails this test, because it is
not a measurement of the effect of pyridostigmine on the final events in the
pathway leading to soman-induced toxicity. Although it is possible that a
useful surrogate could be one that, simply, invariably correlates with the
symptom of interest, and in which the drug’s effect on the surrogate
invariably correlates with, but is not “causally” related to, the desired
clinical effect, reliance on such a surrogate would ordinarily be less
convincing than reliance on a surrogate of the type described in the previous
paragraph.
Indeed, RBC cholinesterase inhibition is not a measure of protection
from soman-induced toxicity at all; as noted above, it is merely reflective
of pyridostigmine plasma levels. A theoretically better surrogate would
be a measure of the degree of pyridostigmine-induced AChE protection
in the face of soman exposure, because this would be a measure of the
proposed mechanism of action. Again, it is critical to recognize that this
mechanism is only proposed, and a correlation of the effect on the
surrogate and the desired effect on the clinical outcome of interest cannot
be considered to constitute proof of the mechanism of disease production
or amelioration.
Ideally, because the life-saving action of pyridostigmine is presumed to be
mediated through its action at the neuromuscular junction (NMJ), evaluation
of the surrogate proposed above should ideally be performed at the NMJ.
However, at least in humans, it is obviously not practical to measure the
treatment effect on the surrogate at the NMJ in all treated patients. One
approach to validating this new surrogate would be to treat animals with
pyridostigmine, expose the animals to soman, and then treat with atropine
and pralidoxime (essentially repeat the dosing regimens in the experiments
previously performed), evaluate the RBC and NMJ for protection of the
enzyme, and demonstrate a correlation between the degree of enzyme
protection in the RBC, enzyme protection at the NMJ, and survival. If this
correlation can be demonstrated, an ex vivo study could be performed in
humans.
Specifically, a small cohort of humans could be treated with an appropriate
regimen of pyridostigmine, followed by blood collection and a muscle biopsy
(preferably of the intercostal muscles, perhaps in a sample of patients
undergoing surgery in which these muscles would ordinarily be exposed).
These tissues could then be exposed to soman ex vivo, and the correlation
between RBC and NMJ enzyme protection could be assessed. If a similar
correlation between blood and NMJ enzyme protection could be

demonstrated in humans as was demonstrated in animals (which would, in

this scenario, have been correlated with increased survival), one could have
greater confidence that the new surrogate (RBC enzyme protection) might
be predictive of human survival. Again, finding such a correlation would not
constitute proof of the effect of pyridostigmine pretreatment in humans, but
it might be considered potentially predictive.
SUMMARY
The preceding discussion is intended to outline the requirements that might

be imposed on any sponsor wishing to bring to market a treatment for
nerve agent poisoning. As can be seen, such an endeavor differs in
important ways from the development of typical treatments; that is,
treatments for naturally occurring diseases which can be adequately
studied. Crucially, the approval of treatments for nerve agent poisoning
depends upon assumptions about mechanisms of disease production, drug
action, and relevance of animal models to humans, considerations that
are usually absent from decisions about drug approval in the typical case.
These considerations may raise new and important (and potentially
unanswerable) questions about the ultimate utility of the treatment in
humans. However, this approach is considered reasonable, given the need
for such treatments and the impossibility of performing the definitive
clinical effectiveness trials.
Indeed, as noted earlier, on February 5, 2003, the application for the
use of pyridostigmine bromide as a pretreatment for soman poisoning, in
conjunction with acute treatment with atropine and pralidoxime was
approved by the FDA. The data provided were considered to have met the
requirements of Subpart I. In particular, the treatment clearly was expected
to provide a meaningful therapeutic benefit, there were a number of
adequate and well-controlled studies in animals, and the animal studies,
taken as a whole, were considered reasonably likely to predict a benefit in
humans.
Specifically, despite a lack of certainty (a situation anticipated by the
rule’s requirement for reasonable likelihood), the mechanism of soman’s
toxicity was considered reasonably well understood, as was the mechanism
of pyridostigmine’s protective effect. Further, the discrepancy in response
across species was considered well explained by the documented relative
differences in carboxylesterase, with the marked increase in protective
ratios produced by carboxylesterase inhibition in pyridostigmine-
pretreated high-carboxylesterase species considered powerful evidence
supporting this theory, and permitting the conclusion that humans will
respond similarly to monkeys.

Finally, the human dose was chosen so as to result in plasma levels shown
to be associated with protection in the monkey. In addition, the resultant
dose was relatively close to a maximum dose considered well tolerated by
otherwise normal, healthy adults.
REFERENCES
1. Federal Food, Drug, and Cosmetic Act, Section 505(d).

2. 21 Code of Federal Regulations 314.500–516, Subpart H.
3. 21 Code of Federal Regulations 314.500–516, Subpart H.
4. Federal Register, Vol. 64, No. 162, October 5, 1999, 53960–53970.
5. Federal Register, Vol. 64, No. 162, October 5, 1999, 53960–53970.
6. Evison, D.; Hinsley, D.; Rice, P. Chemical Weapons. BMJ 2002, 324, 332–335.
7. Gunderson, C.H., et al. Nerve Agents: A Review. Neurology 1992, 42, 946–
950.
8. Abramowicz, M., Ed.; Prevention and Treatment of Injury From Chemical
Warfare Agents. The Medical Letter 2002, 44, 1121.
9. Golomb, B.A. A Review of the Scientific Literature As It Pertains to Gulf War
Illnesses. Pyridostigmine Bromide. Santa Monica: Rand, 1999; 11–48.
10. Sidell, F.R.; Borak, J. Chemical Warfare Agents: II. Nerve Agents. Annals of
Emergency Medicine 1992, 27, 7, 865–871.
11. Harris, L.W., et al. Apparent Relationship Between Decarbamylation Half-Time
and Efficacy Against Soman Lethality In Different Species. The Pharmacologist
1985, 27 (3), 134.
12. Ellin, R.I.; Kaminskis, A. Carbamoylated Enzyme Reversal as a Means of
Predicting Pyridostigmine Protection Against Soman. J. Pharm. Pharmacology
1989, 41, 633–635.
13. Wetherell, J.R.; French, M.C. A Comparison of the Decarbamoylation Rates of
Physostigmine-Inhibited Plasma and Red Cell Cholinesterases of Man with Other
Species. Biochemical Pharmacology 1991, 42 (3), 515–520.
14. Maxwell, D.M., et al. Effect of Carboxylesterase Inhibition on Carbamate
Protection Against Soman Toxicity. The Journal of Pharmacology and
Experimental Therapeutics 1988, 246, 986–991.
15. Maxwell, D.M., et al. Comparison of Antidote Protection against Soman by
Pyridostigmine, Hl-6 and Acetylcholinesterase. The Journal of Pharmacology
and Experimental Therapeutics 1993, 264, 1085–1089.
16. Raveh, L., et al. The Involvement of the NMD A Receptor Complex in the
Protective Effect of Anticholinergic Drugs Against Soman Poisoning. Neuro
Toxicology 1999, 20 (4), 551–560.
17. Carpentier, P., et al. Effects of Thienylphencyclidine (TCP) on Seizure Activity
and Brain Damage Produced by Soman in Guinea-Pigs: EcoG Correlates of
Neurotoxicity. Neuro Toxicology 2001, 22, 13–28.

18. Lallement, G., et al. Review of the Value of Gacyclidine (GK-11) as Adjuvant
Medication to Conventional Treatments of Organophosphate Poisoning: Primate
Experiments Mimicking Various Scenarios of Military or Terrorist Attack by
Soman. Neuro Toxicology 1999, 20 (4), 675–684.
19. Svensson, I., et al. Soman-Induced Interleukin-1 Beta mRNA and Protein in Rat
Brain. Neuro Toxicology 2001, 22, 355–362.
20. Fleming, T.R.; DeMets, D.L. Surrogate End Points in Clinical Trials: Are We
Being Misled? Ann Intern Med 1996, 125, 605–613.

25
Bioequivalence Assessment:
Approaches, Designs, and Statistical
Considerations
Rabindra N.Patnaik*
INTRODUCTION
Bioavailability (BA) and bioequivalence (BE) are very closely related.

Bioavailability usually focuses on the release of the active ingredient/active
moiety from the drug product to one or more sites of action. Bioequivalence
focuses primarily on the comparison of the measures of release of the active
moiety (drug substance) between two (test and reference) products. Studies
based on BE principles are useful during drug development and approval of
a new chemical entity drug product during the IND/NDA period to link
between various formulations, to examine the effect of various factors on
BA of the drug, and to study the pharmacokinetics of the drug. Bioavailability
and bioequivalence principles have been discussed exten-sively in Chapters
2, 9, 17, and 19 of this book. Bioequivalence assessment is a dynamic and
evolving discipline with complexities. It has evolved significantly during the
* Current affiliation: Watson Laboratories, Inc., Corona, California, U.S.A.
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562 Patnaik
past few years. Significant information on this rapidly evolving field may be
found in the literature [1–10].
The discussion in this chapter is limited to data analysis of BE studies and
in no way comprehensive. It focuses on BE studies with a pharmacokinetic
endpoint (systemic exposure approach) with emphasis on the practical aspects
of bioequivalence assessment from a nonstatistician standpoint.
APPROACHES TO BE ASSESSMENT
It is generally acceptable that differences between formulations would be

observed due to biological and other variabilities. Thus, it is important to
examine the clinical relevance of such observed/estimated differences and to
ascertain how much difference would be acceptable from the safety and
efficacy standpoint. Furthermore, it is equally important to ascertain the
degree of uncertainty from such a study and also the magnitude of uncertainty
that would be acceptable if a difference is observed. In order to address these
issues/questions, statistical principles and methodologies are applied.
Assessment of BE is a dynamic field in the biopharmaceutic evaluation of
product quality. Various approaches have been proposed to assess BE. These
are (a) average bioequivalence (ABE) and (b) population bioequivalence (PBE)
and individual bioequivalence (IBE). Each approach has various advantages
and disadvantages. However, ABE is the generally applied approach as it is
widely acceptable to the regulatory authorities for the approval of drug
products. For completeness, PBE and IBE approaches are briefly described,
but the focus in this chapter will be on ABE. A brief description of various
approaches is presented below:
Average Bioequivalence
Average bioequivalence compares the population averages between the test
and reference products. It is based on the ratio of average bioavailability
measures of the test and reference formulations over all individual subjects/
patients in the study population. The details of the ABE criteria have been
described [11].
Population and Individual Bioequivalence

These are novel approaches that include comparison of both population
means and variances (variability). In theory, the PBE and IBE approaches
reflect different objectives of BE testing that may be conducted at various
stages of drug development. These PBE and IBE objectives are embodied in
the concepts of prescribability and switchability, respectively [11–15]. In

Bioequivalence Assessment 563
addition to the population means, while the PBE approach assesses total
variability of the BE metrics, the IBE approach focuses on intraindividual
variability of the test and reference products, as well as subject-by-formulation
interaction. These factors are important considerations for interchangeability
of a drug product. In addition, both PBE and IBE criteria can be scaled to the
reference variability. This offers an advantage for BE assessment of certain
drug products, i.e., highly variable drugs [11]. There has been considerable
debate in the literature about the utility of these approaches and these
approaches have not been adopted for BE assessment by any regulatory
authorities.
CONSIDERATIONS FOR BIOEQUIVALENCE STUDIES
BE Study Designs
Various study designs are available for conducting a BE study. The design
depends on the pharmacokinetics of the drug, and the number and type of
treatments to be tested. Discussions on designs can be found in the literature
(16–27).
Crossover Design
In most cases, a crossover design is preferred. In this design, each subject
acts as his/her own control, thus minimizing the variability and increasing
the study power. Examples of various crossover designs are presented in
Table 2. The following are the two major classes of crossover designs that
are used in BE studies.
Nonreplicated Crossover Design. A standard two-treatment, two-period,
two-sequence design study is an example of this design. In this design, each
treatment is administered no more than once to each subject. Basically, one
half of the subjects/patients are treated with one drug (test drug) and the
other half is treated with the second drug (comparator or reference drug)
during the first period. After an adequate washout period (time required for
complete elimination of the drug from the system based on the elimination
half-life of the drug), each group of subjects is switched to the other drug in
the second period.
In a nonreplicated crossover study other than the standard two-treatment,
two-period study, the number of sequences appropriate for the study depends
on the number of drug products (treatments) under study. It is usually a
good practice that the study design be completely balanced for sequences
with respect to the number of subjects.

564 Patnaik
TABLE 1 Examples of Dosage Forms for Bioequivalence Assessment
Replicated Crossover Design. Examples of replicated crossover design

are presented in Table 2. In this design some of the subjects receive at least
one of the treatments more than once. In a standard replicated crossover
design study, a single batch or a lot of each of the drug products is dosed
twice to the same subject. Each treatment is separated by a washout period.
Recently much attention has been focused on replicate design studies
because of their ability to identify subject by treatment interactions (10–15).
Replicated single-dose crossover studies have been recommended for
modified-release products such as, extended-release dosage forms and
transdermal systems that may have different drug-release mechanisms.
Furthermore, replicate design studies are often recommended for highly
variable drugs where a large number of subjects/patients are needed to achieve
adequate study power to demonstrate BE. These study designs are also useful
for examining intrasubject variability associated with different treatments,
presence and magnitude of subject-by-formulation interactions, and unequal
carryover effect of the treatments.
Generally a two-treatment, two-sequence, four-period, replicated crossover
design has been used for a BE study while using average BE, individual BE or
population BE approaches [11]. However, for regulatory decision making,

Bioequivalence Assessment
TABLE 2 Some Examples of Nonreplicated and Replicated Study Designs
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566 Patnaik
even if a design of BE study is replicate, the acceptable statistical data analyses

has to be based on ABE.
Parallel Design
In some cases, such as long-half-life drugs and cytotoxic drugs, there is a
concern for using a crossover design. For a long-half-life drug, crossover
designs are difficult to conduct, as the study would take a very long time to
complete. There is also a strong likelihood of significant subject dropouts,
creating problems with study power. In the case of cytotoxic drugs, it is not
ethical or appropriate to unnecessarily expose the individuals to the toxic
compounds twice. As a result, a parallel design where each group of
individuals is simultaneously dosed with a treatment only once is often
recommended. However, this design would generally require more subjects
than would be required for a crossover design because of inadequate study
power considerations.
Sequential Design
Sequential designs are increasingly being used in major clinical trials

concerning life-threatening diseases. Most applications have trials designed
to establish whether an experimental treatment is superior to a control.
However, many trials are conducted with the objective of showing that an
experimental treatment is equivalent to a control. Methods have been
developed in the context of bioequivalence and appropriate sequential
procedures are identified [25–27]. The likelihood of demonstrating
bioequivalence when the formulations are truly equivalent depends on the
sample size and on the variability of the bioequivalence endpoint. Group
sequential bioequivalence testing provides a statistically valid way to
accommodate misspecification of variability in designing the trial by allowing
for additional observations (which have to be prespecified in the protocol) if
a clear decision to accept or reject bioequivalence cannot be reached with
the initial set of observation [27].
Study Population
Usually, subjects recruited for in vivo BE studies should be 18 years of age or

older and capable of giving informed consent. Bioequivalence studies may
be conducted on healthy populations or target (patient) populations
depending on the type of drug under study. It is recommended that in vivo
BE studies be conducted in individuals representative of the general
population, taking age, gender, and race factors into account. If the drug

product is intended for use in both genders, attempt should be made to include
equal numbers of males and females in the study. If the drug product is to be
used predominantly in the elderly, attempt should be made to include as
many subjects 60 years of age or older as possible. The total number of
subjects in the study should provide adequate power for BE demonstration,
but it is not expected that there will be sufficient power to draw equivalence
conclusions for each subgroup. In such cases, statistical analysis of subgroups
is not recommended. Restrictions on admission into the study should generally
be based solely on safety considerations. In some instances, it may be useful
or even necessary that BE study subjects consisting of the target population
for the specific drug. In this situation, attempt should be made to enroll
patients whose disease process would be stable for the duration of the BE
study.
For the subject selection, inclusion and exclusion criteria should be well
defined. Medical history, physical examination, clinical evaluation, and all
restrictions (inclusion and exclusion criteria) prior to and during the conduct
of the study should be well defined in the protocol and should be strictly
adhered to.
Sample Size
This is one of the most important considerations in the assessment of BE.
There are important issues to consider while developing a protocol for a BE
study, such as (a) how much of a chance or probability of concluding
equivalence is desired? (b) what true ratio of test/reference (T/R) averages is
one interested in?, and (c) what is the anticipated within-subject coefficient
of variation (CV) of the BE metrics?
While developing a protocol for an in vivo BE study, a sufficient number
of subjects should be enrolled to achieve adequate study power. Attention
should be paid to the possibility of dropouts, add-on subjects, individuals or
groups, and replacement subjects. The enrolled subjects should be completely
randomized for treatments and sequences. Attempts should be made to assign
the same number of subjects to each sequence to make the study balanced. If
a multi-center/site/group study is planned, an adequate number of subjects
should be enrolled at each site or in each group.
Generally, a minimum number of twelve evaluative subjects may be
included in any BE study [11]. When an average BE approach is selected
using either nonreplicated or replicated designs, methods appropriate to the
study design should be used to estimate sample sizes. Sample sizes for average
BE may be obtained using published formulas. The study should have 80 or
90% power to conclude BE between the formulations. Sample size also
depends on the magnitude of variability and the design of the study. Variance
estimates to determine the number of subjects for a specific drug can be

568 Patnaik
obtained from the literature and/or pilot studies. Information on sample size
can be found in the literature [28–33].
A sufficient number of subjects should be enrolled in the study to allow
for dropouts. Because replacement of subjects during the study could
complicate the statistical model and analysis, dropouts generally should not
be replaced. If dropouts are to be replaced during the study, the intention
should be stated in the protocol a priori. The protocol should also state
whether samples from replacement subjects would be analyzed even if their
data would not be included in the statistical analysis. If the dropout rate is
high and sponsors wish to add more subjects, a modification of the statistical
analysis may be needed. Additional subjects should not be included after
data analysis unless the trial was designed from the beginning as a group
sequential design.
BIOEQUIVALENCE CRITERIA
The average BE approach is used to assess bioequivalence for all drug

products. Thus, the discussion is predominantly focused on the average BE
approach. However, other approaches and criteria have been developed in
the recent years. Information on these new approaches and the associated
methodologies are available in the literature [11–15].
The general form of the average BE criteria is presented below:
where:
µT—population mean for the test product
µR—population mean for the reference product
θA1—lower limit of the confidence interval (0.80)
θA2—upper limit of the confidence interval (1.25)
Analysis of BE data using the average BE approach focuses first on

estimations of the mean difference between test and reference products for
the log-transformed BA measure. Subsequently, the general approach is to
construct a 90% confidence interval for the difference in the population
means and to reach a conclusion of average BE if this confidence interval is
contained in the interval. Due to the nature of normal-theory confidence
intervals, this is equivalent to carrying out two one-sided tests of hypothesis
at the 5% level of significance [34]. The 90% confidence interval for the
difference in the means of the log-transformed data is calculated using
methods appropriate to the experimental design. The antilogarithm of the
confidence limits constitutes the 90% confidence interval for the ratio of the
geometric means between the test and reference products.

STATISTICAL METHODS AND DATA ANALYSIS
Data Processing
Analyses of BE data are typically based on a statistical model for the logarithm
of the BE measures. The BE measures are log- transformed generally using
natural logarithms. Common logarithms to the base 10 may be used. The
choice of natural or common logarithm should be consistent and should be
stated in the study report. The limited sample size in a typical BE study
precludes a reliable determination of the distribution of the data set. It is not
necessary to test for normality of error distribution after logtransformation,
or to use normality of error distribution as a reason for carrying out the
statistical analysis on the original scale [35]. Logarithm transformation is
universally accepted by the national and international biopharmaceutic
scientific community and regulatory authorities. However, if there is a need
to use data on the original scale, adequate justification should be documented
and provided in the study report.
Statistical Methods and Data Analysis

The following discussion focuses on the statistical methods and analysis of
data pertaining to the assessment of BE by applying the average BE approach
and criteria and using both nonreplicated and replicated crossover study
designs. The statistical methods applied to the individual and population BE
approach are beyond the scope of this discussion and are not included in this
chapter.
Nonreplicated Crossover Designs. For analysis of variance (ANOVA),
general linear model procedures (PROC GLM) available in SAS, or equivalent
software may be used, although linear mixed-effects model procedures may
also be used for analysis of nonreplicated crossover studies.
For example, for a conventional two-treatment, two-period,
twosequence (2×2) randomized crossover design, the statistical model
typically includes factors accounting for the following sources of variation:
sequence, subjects nested within sequences, period, and treatment. The
ESTIMATE statement in SAS PROC GLM, or equivalent statement in
other software, is used to obtain estimates for the adjusted differences
between treatment means and the standard error associated with these
differences. A simple example of the codes using SAS version 6 12 for a
conventional twotreatment, two-period, two-sequence crossover BE study
are presented below:
PROC GLM DATA=EXAMPLE;
CLASS SUBJ SEQ PER TRT;

570 Patnaik
MODEL LAUCT LAUCI LCMAX=SEQ SUBJ(SEQ) PER TRT;

TEST H=SEQ E=SUBJ(SEQ)/HTYPE=3 ETYPE=3;
ESTIMATE “A vs. B” TRT 1–1;
LSMEAN TRT;
RUN;
where:
SUBJ=subject
SEQ=sequence or order of drug administration
PER=period or phase of drug administration
TRT=treatment or formulation or product (A=test, B=reference)
LAUCT=log(AUC0-t)
LAUCI=log(AUC0-inf)
LCMAX=log(CMAX)
In the case of a nonreplicated crossover design, only one “MODEL”
statement is used for all BE measures in ANOVA. The “TEST” statement
examines the sequence effect (statistically significant, if p<0.1). The output
of results using a simulated data set and the above codes as an example is
presented in Table 3.
The “ESTIMATE” statement pertains to a two- treatment study design in
which the code “A” (test product) precedes “B” (reference product). If the
treatments were changed to “T” (test product) and “R” (reference product);
the “ESTIMATE” statement would be changed to:
ESTIMATE T vs. R’ trt—1 1;
since “R” precedes “T” in the alpha numeric sort order.
Furthermore if there are three treatments, for example, “A” (test product
1), “B” (test product 2), and “C” (reference product), the “ESTIMATE”
statements may be changed as follows to estimate differences between
products:
ESTIMATE ‘A vs. B’ trt 1–1 0; (Difference between trt A and trt B)
ESTIMATE ‘A vs. C trt 1 0–1; (difference between trt A and trt C)
ESTIMATE ‘B vs. C trt 0 1–1; (difference between trt B and trt C)
Replicated Crossover Design. Linear mixed-effects model procedures,
available in PROC MIXED in SAS or equivalent software, may be used for
the analysis of replicated crossover studies for average BE. The ESTIMATE
statement in SAS PROC MIXED, or equivalent statement in other software,
is used to obtain estimates for the adjusted differences between treatment
means and the standard error associated with these differences. An example
of SAS code (version 6.12) statements is presented below [11]:
PROC MIXED DATA=EXAMPLE;
CLASSES SEQ SUBJ PER TRT;

Bioequivalence Assessment
TABLE 3 The GLM (General Linear Models) Procedure
571
572 Patnaik
MODEL Y=SEQ PER TRT/DDFM=SATTERTH;

RANDOM TRT/TYPE=FA0(2) SUB=SUBJ G;
REPEATED/GRP=TRT SUB=SUBJ;
ESTIMATE ‘A vs. B’ TRT 1–1/CL ALPHA=0.1;
RUN;
where:
SUBJ=subject
SEQ=sequence or order of drug administration
PER=period or phase of drug administration
TRT=treatment or formulation or product (A=test, B=reference)
Y=LAUCT=log(AUC0-t) or LAUCI=log(AUC0-inf) or LCMAX=log(CMAX)
For analyzing a data set from a replicated crossover design, each BE
measure (AUC, CMAX, etc.) is analyzed separately using the above set of
SAS codes. Thus the “MODEL” statement specifies one BE measure at a
time. An advantage of using a replicated crossover design is that it is possible
to determine the estimates of variances associated with betweensubject and
within-subject for test and reference products and subject-byformulation
interaction. The resultant output from the analysis of a simulated data set as
an example from a two-treatment, two-sequence, fourperiod replicated
crossover design study is presented in Table 4.
Parallel Design. For parallel designs, analysis of variance using general
linear model procedures available in SAS PROC GLM or equivalent software
may be used. The statistical model typically includes a factor accounting for
only one source of variation—treatment. There are no sources of variation
associated with sequence or period as there are no sequences or periods in a
parallel design.
PROC GLM DATA=EXAMPLE;
CLASS SUBJ TRT;
MODEL LAUCT LAUCI LCMAX=TRT;
ESTIMATE ‘A vs. B’ TRT 1–1;
LSMEAN TRT;
RUN;
The confidence interval for the difference of means in the log scale can be
computed using the total between-subject variance.
Estimation of 90% Confidence Interval

Average BE assessment is carried out by determining the 90% confidence
interval of the estimate of the difference between the logarithm-transformed
means of test and reference BE measures using the two one-sided tests

TABLE 4

574 Patnaik
TABLE 4 Continued

TABLE 4 Continued
procedure [11,34]. The general expression for the test procedure is presented
below:
(µT-µR)±tedf (0.95)*SE
or,
(µT-µR)+tedf (0.95)*SE (upper confidence limit)
(µT-µR)-tedf (0.95)*SE (lower confidence limit)
or
Estimate+t0.95(edf)*SE (upper confidence limit)
Estimate-t0.95(edf)*SE (lower confidence limit)
where:
µT—population mean for test product
µR—population mean for reference product
t0.95(edf)—95th percentile of the Student’s t-distribution for error degrees of
freedom from t distribution table (p=0.05) or computed from various software
packages
edf—degrees of freedom associated with the error term in the result output
from the PROC GLM statements

576 Patnaik
Estimate—estimate of the difference between test and reference means

(geometric) (from the output of the ESTIMATE statement of the PROC GLM
code)
SE—standard error of the estimate of the difference between the test and
reference product means (from the output of the ESTI-MATE statement of
the PROC GLM code)
The antilogarithm of the ESTIMATE gives the test/reference ratio in the
normal scale, and the antilogarithm of the confidence limits constitute the
90% confidence interval for the ratio of the geometric means between the
test and reference products. An example of the computation of the 90%
confidence interval with illustration is presented in Table 5.
The SAS code for the PROC GLM procedure with the “ESTIMATE”
statement (at least as of SAS version 6.12) presented above for the
nonreplicated crossover design would not calculate the 90% confidence
interval. Alternate PROC GLM statements shown below would estimate the
90% confidence interval:
PROC GLM DATA = EXAMPLE;
CLASS SUBJ SEQ PER TRT;
MODEL LAUCT LAUCI LCMAX = SEQ SUBJ(SEQ) PER TRT;
TABLE 5 Example of Estimation of 90% Confidence Interval using Two One-sided

t-tests (Estimates and other data taken from Table 3)

TEST H = SEQ E = SUBJ(SEQ)/HTYPE = 3 ETYPE = 3;

LSMEANS TRT/PDIFF CL ALPHA = 0.10;
RUN;
There may be some disadvantages in using the “LSMEANS” statement
instead of the “ESTIMATE” statement. “LSMEANS” may calculate an
erroneous difference depending on how the treatments are coded (e.g., R-T
instead of T-R). This is due to the alphanumeric sort order used by the SAS.
Furthermore, in some cases LSMEANS would be “nonestimable”, but the
difference between the LSMEANS would be estimable. The “ESTIMATE”
statement would give the estimate of the difference between the test and
reference least squares means and thus the 90% confidence interval could be
estimated. Unlike the PROC GLM procedure, the “ESTIMATE” statement
in PROC MIXED procedure shown above for the replicated crossover design
would estimate the 90% confidence interval.
ADDITIONAL ISSUES RELATED TO BE STUDY
Add-on Subjects and Group Effect
The BE study protocol should consider the following important factors:
i. appropriate subject inclusion and exclusion criteria,

ii. enrollment of more than the required number of subjects to achieve
adequate study power and to compensate for any unexpected
dropouts,
iii. randomization of all subjects as a single group before starting the
study,
iv. analysis of all study samples at a single analytical site,
v. statistical analysis of data on the BE measures as a single data set.
Generally, BE study designs with add-on subjects are not recommended.

However, on many occasions, add-on subjects (groups) are used in a crossover
study for a variety of reasons. Examples of study designs with add-on subjects
are presented in Table 6. Additional subjects may be enrolled either
overlapping the periods of the study or greatly separated in time. Sometimes
studies are also conducted at multiple centers, thus additional subjects may
be enrolled at different sites at different times. In other cases, for logistical
reasons only a limited number of subjects can be studied at one time at a
single site, thus creating different groups of subjects.
It is emphasized that there may be considerable risk in using add-on subjects
as a discrete group to increase the power of the study after the study has
been completed. Using add-on subjects would be like a “second” study with

578 Patnaik
TABLE 6 Examples of Study Designs with Different Groups
a different group of subjects. Thus, statistical analysis would be carried out

to examine whether these two groups of subjects responded differently to
the test and reference products (group by treatment interaction). If a
statistically significant interaction is detected, it is possible that the data from
the two groups cannot be combined to establish bioequivalence. Under those
circumstances, the statistical model should be modified to reflect the
multigroup nature of the study. In particular, the model should reflect the
presence of different groups and the fact that the periods for the first group
are different from the periods for the second group. Sometimes the study is
carried out in two or more groups and those groups are studied at different
clinical sites, or at the same site but separated in time (for example, months
apart). Questions may arise as to whether the results from the several groups
should be combined in a single analysis.
If one decides to use an add-on study design, the following procedures
may be considered:
i. All plasma samples from the two groups should be analyzed at

one time. The samples of each subject from two periods should
be analyzed together.

ii. Subjects should be randomized and balanced for treatments and

sequences. Thus, in the add-on group there should be an EQUAL
number of subjects in each of the two treatment administration
sequences (test followed by reference, and reference followed by
test), or as close to equal as possible if the number of subjects
recruited for the “second study” is an odd number.
iii. The statistical model used to analyze the data should reflect the
fact that periods 1 and 2 in the “add-on study” are not the same
as periods 1 and 2 in the initial study.
iv. Group by treatment interaction should be examined by
appropriate statistical analysis if it is considered that there are
two discrete groups. If group by treatment interaction is
statistically significant (at the 0.10 level of significance), data from
the two groups may or may not be combined depending on several
factors. If statistically significant interaction is not detected, this
term (the group by interaction term) may be dropped from the
statistical model used to compute the 90% confidence interval.
Some examples of the statistical model (in SAS code, version 6.12) for the
analysis of variance (ANOVA) to examine the group effect are presented
below:
CLASS GRP SUBJ SEQ PER TRT;
MODEL LAUCT LAUCI LCMAX =
GRP
SEQ
SUBJ(SEQ)
PER(GRP)
TRT
GRP*TRT;
TEST H = SEQ E = SUBJ(SEQ)/HTYPE = 3 ETYPE = 3;
LSMEAN TRT;
RUN;
An extensive statistical model may be:
GRP
SEQ

580 Patnaik
GRP*SEQ
SUBJ(SEQ*GRP)
PER(GRP)
TRT
GRP*TRT;
TEST H = GRP E = SUBJ(SEQ*GRP)/HTPE = 3 ETYPE = 3;
TEST H = SEQ*GRP E = SUBJ(SEQ*GRP)/HTYPE = 3
ETYPE = 3;
TEST H = SEQ E = SUBJ(SEQ*GRP)/HTYPE = 3 ETYPE = 3;
LSMEAN TRT;
RUN;
where:
GRP = group
SUBJ = subject
PER = period
SEQ = sequence
TRT = treatment
The “TEST” statements in the second model examine the statistical
significance of GROUP, SEQ*GROUP, and SEQ effects (statistically
significant if p < 0.1). These are supportive information regarding the study.
However, GROUP*TRT is the important source of variance.
In the event that GROUP*TRT interaction is not statistically significant
(p>0.1), this term may be dropped from the model and the data reanalyzed
for BE assessment (estimation of confidence interval). An example of SAS
code without the GROUP*TRT term in the model is presented below:
SEQ
SUBJ(SEQ)
PER(GRP)
TRT;
TEST H = SEQ E = SUBJ(SEQ)/HTPE = 3 ETYPE = 3;
LSMEAN TRT;
RUN;
On the other hand, if a statistically significant GRP*TRT effect is observed
(p<0.1), careful consideration should be given as to the appropriateness of
combining the data from the two groups. If data should not be combined,
BE may be assessed using data from the original/(first) group only.

Group sequential design, in which the decision to study a second group of

subjects is based on the outcome from the first group, calls for different
statistical methods and is outside the scope of this discussion. However, the
discussions on this design can be found in the literature [25–27].
Outliers
Discussions regarding the outlier issue in BE assessment can be found in

literature [36, 37]. On many occasions, discordant or “abnormal” response
to the administered treatment is observed for certain subjects as compared
to the rest of the study population. There is always a strong tendency to
drop those subjects from the data set for BE assessment.
Abnormal response may be considered into the following categories:
Pharmacokinetic Anomaly
This pertains to an unusual value(s) in the drug level in the biological fluid
that does not conform to the predicted pharmacokinetic response of that
subject at that sampling time. Sometimes the common practice is to reassay
only the specific sample(s) in question to confirm the original value or, if
appropriate, substitute the original value with the new value generated
from the original and reassay values as per the SOP. However, in order to
document the reproducibility of the original assay values, it may be prudent
to reassay the “suspect” sample(s) along with the “normal” samples of
that subject from adjoining sampling times, both earlier and later.
Alternatively, plasma samples from the entire treatment for that subject
may be reassayed to decide on the substitution of the suspect data. The
specific procedure(s) to be followed for the disposition of the
pharmacokinetic anomaly must be decided a priori.
Aberrant BE Measure
This is one of the important issues in the assessment of BE. On certain
occasions, subject data for one or more BE measures exhibit “suspect”
discordant values compared to the rest of the subjects in a study. Because BE
studies are usually carried out as crossover studies, the most important type
of subject outlier is the within-subject outlier, where one subject or even a
few subjects differ notably from the rest of the subjects with respect to a
within-subject test-reference comparison. The existence of a subject outlier
with no protocol violations could be a manifestation of subject-by-
formulation interactions or product failure.

582 Patnaik
There may be a tendency to drop the discordant data from the statistical
analysis without understanding the probable origin of an aberrant response
of that subject. The deletion of that discordant data may have significant
impact on the outcome of the study. The clinical protocol for the specific
subject(s) may be extensively examined for any protocol violation. In addition,
the product quality, such as content uniformity or homogeneity of the testing
batch, and the dissolution properties of the product batch in question may
be examined as possible cause(s) of this discordant behavior. If no probable
cause can be ascertained, the discordant subject is often redosed with a few
other study subjects who showed normal response, and their response is
redocumented. If the original data were reproduced for the discordant subject,
it would show that the original data represent the true response and the
subject should not be deleted from the data set. On the other hand, if the
redosed data conform to the response of the other subjects in the original
study with the observed intersubject variability, it would show that the
unexplainable discordant value probably originated at random and there is
probably good reason that the discordant data may be dropped from the
original data set.
Statistical Outlier
In the past, statistical tests were often applied to identify statistical outliers
in the data set. Based on those tests, it was a common practice to drop the
discordant data from the data set used for statistical analysis and estimation
of the confidence interval. This approach may be unacceptable to some
regulatory authorities as it may be due to an underlying, although unidentified
reason, instead of being a random occurrence.
Carryover Effect
Carryover (residual) effect is the influence of one treatment administered in
a particular period on the response to a treatment in the subsequent period
of the study design. Use of crossover designs for BE studies allows each
subject to serve as his or her own control to improve the precision of the
comparison. One of the assumptions underlying this principle is that carryover
effects are either absent or equal for each formulation and preceding
formulation. In BE studies it is generally assumed that one only needs to
consider first-order carryover effect, i.e., effects that a treatment has on the
response to a treatment administered in the next period. However, it is also
important to consider the possibility that the carryover effect depends not
only on the preceding treatment but also on the treatment being preceded.
This is called Direct-by-Carryover interaction. If carryover effects are not
equal, then the estimate of difference between the treatment means that is

obtained without the carryover effects in the model may be biased. The need
to consider more than just simple first-order carryover effects have been
emphasized [38].
In a standard two-formulation, two-period, two-sequence nonreplicated
crossover design, the sequence test in the analysis of variance is only available
to test for the presence of unequal carryover effects. However, this is a
between-subject test, which would be expected to have poor discriminating
power in a typical BE study. Furthermore, if the possibility of unequal
carryover effects cannot be ruled out, an unbiased estimate of the difference
between the test and reference means based on within-subject comparisons
cannot be obtained with this design [11].
For most cases of both replicated and nonreplicated crossover designs,
the possibility of unequal carryover effects is considered unlikely in a BE
study under the following circumstances [17]:
1. The study is single-dose.

2. The drug is not an endogenous entity.
3. An adequate washout period has been allowed between periods
of the study, and in the subsequent periods the predose biological
matrix samples do not exhibit a detectable drug level in any of
the subjects.
4. The study meets all scientific criteria (e.g., it is based on an
acceptable study protocol and the matrix samples were assayed
using a fully validated methodology).
With respect to a multiple-dose study, it is believed that the possibility of an

unequal carryover effect may also be discounted, provided the drug is not an
endogenous entity and the study meets all scientific criteria as described above.
Under all other circumstances, it is prudent to consider the possibility of
unequal carryover effects, including a direct-by-carryover effect.
SUMMARY AND CONCLUSION

Bioequivalence is an evolving applied discipline with various complexities.
There have been significant developments in the area of BE assessment in
recent years. However, there are several unresolved issues. Novel dosage
forms are being developed that will require novel approaches to assess BE.
The current approaches and methods applied to the assessment of BE are
scientifically sound and dependable. However, due to the limited size of a
typical study, every effort should be made to conduct the study with a
wellplanned objective and protocol, and established methodologies and
controls, so that unbiased data will be obtained to yield reliable results. As a
result, conclusions derived from these studies will be scientifically valid and

584 Patnaik
reliable. Pharmaceutical scientists and statisticians are making continuous

efforts to improve existing methodologies and to develop new methodologies
that will possibly require fewer resources and will reduce human testing.
ACKNOWLEDGMENT
I acknowledge the valuable help and suggestions of Huaixiang Li, Ph.D. for
the statistical discussions and Wallace P.Adams, Ph.D. in preparing this article.
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